Acetolactate synthase having 2-ketoisovalerate decarboxylase activity and uses thereof

ABSTRACT

Provided herein are metabolically-modified microorganisms useful for producing biofuels. More specifically, provided herein are methods of producing high alcohols including isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol from a suitable substrate and a recombinant acetolactate synthase having both synthase and decarboxylase activity.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/095,830, filed Apr. 27, 2011, now U.S. Pat. No. 9,193,965, whichclaims the benefit of U.S. Provisional Application No. 61/328,327, filedApr. 27, 2010, the disclosures of which are incorporated herein byreference.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 54626_SEQ_FINAL.txt. The text file is 63.5 KB;was created on Nov. 11, 2015; and is being submitted via EFS-Web withthe filing of the specification.

TECHNICAL FIELD

The invention relates to polypeptides having 2-ketoisovaleratedecarboxylase activity. More particularly, the invention providesrecombinant polypeptides having 2-ketoisovalerate activity andbiosynthetic pathways having such activity.

BACKGROUND

Artificial or recombinant biosynthetic pathways are useful in thegeneration of novel byproducts or in the generation of existing productsby a new pathway. Enzymes are key players in the production of newbyproducts or in modulating biosynthetic pathways.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

The disclosure provides a recombinant microorganism that producesisobutanol wherein the alcohol is produced from a metabolite comprising2-keto acid and wherein the organism lacks a gene encoding 2-keto aciddecarboxylase.

The disclosure also provides a recombinant microorganism that producesisobutanol wherein the alcohol is produced from a metabolite comprising2-keto acid and wherein the organism expresses a heterologousacetolactate synthase having decarboxylase activity.

The disclosure further provides a recombinant microorganism thatproduces isobutanol wherein the alcohol is produced from a metabolitecomprising 2-keto acid and wherein the organism comprises a heterologousmutant acetolactate synthase lacking 2-keto acid decarboxylase activityand a heterologous 2-keto acid decarboxylase.

In certain embodiments of the foregoing, the microorganism is anEschirichia coli. In some embodiments, the acetolactate synthase isderived from Baccilus subtilis. In yet other embodiments, themicroorganism is selected from a genus of Corynebacterium, Bacillus,Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus,Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia,Shigella, Klebsiella, Citrobacter, Saccharomyces, Dekkera, Klyveromyces,and Pichia. In still further embodiments, the biosynthetic pathway forthe production of an amino acid in the organism is modified forproduction of isobutanol. In some embodiments, the microorganismcomprises reduced ethanol production capability compared to a parentalmicroorganism. In certain embodiments, the acetolactate synthasecomprises a sequence that is at least 80%, 90%, 95%, 98%, 99% or 100%identical to SEQ ID NO:2 and has acetolactate synthase and decarboxylaseactivity. In yet other embodiments, the acetolactate synthase comprisesa sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQID NO:7 and has acetolactate synthase activity and the organism furthercomprises a 2-keto acid decarboxylase.

The disclosure also provides a method for producing isobutanol, themethod comprising culturing a microorganism of the disclosure in thepresence of a suitable substrate or metabolic intermediate and underconditions suitable for the conversion of the substrate or metabolicintermediate to isobutanol, and substantially purifying the isobutanol.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIGS. 1A-1C show a schematic representation of the pathway forisobutanol production. FIG. 1A: The Kdc-dependent synthetic pathway forisobutanol production. FIG. 1B: Isobutanol production with theKdc-dependent and -independent synthetic pathways. IlvC, acetohydroxyacid isomeroreductase; IlvD, dihydroxy acid dehydratase. FIG. 1C:Enzymatic reaction of Als, Ahbs, and Kdc activities.

FIGS. 2A-C shows a summary of results for isobutanol production withoutKdc in E. coli. The cells were grown in M9 medium containing 5 g/literyeast extract and 36 g/liter glucose in shake flasks at 30° C. with 0.1mM IPTG for 24 hrs. Over-expressed and deleted genes and KIVsupplementation are indicated below the graphs. FIG. 2A: Isobutanolproduction using various enzymes. FIGS. 2B and 2C: Isobutanol productionwith the supply of KIV. ND, not detectable.

FIG. 3A shows the active site region of K. pneumoniae AlsS. Q483 and TPPreacted with the first pyruvate are marked. FIGS. 3B and 3C: Isobutanolproduction with the AlsS mutants.

FIGS. 4A-4C show the effect of Q487 on the decarboxylase activity ofAlsS. FIG. 4A: Isobutanol production with the AlsS variants. The cellswere grown in M9 medium containing 5 g/liter yeast extract and 36g/liter glucose in shake flasks at 30° C. with 0.1 mM IPTG for 24 hrs.FIGS. 4B and 4C: Specific growth rate of E. coli strain KS145 (ΔilvIΔilvB) with the AlsS variants in M9 medium containing 1% glucose and39.5 μg≠ml⁻¹ L-isoleucine (FIG. 4B) and 35 μg·ml⁻¹ L-valine and 39.5μg·ml⁻¹ L-leucine (FIG. 4C). The ilvC and ilvD genes were over-expressedwith the alsS gene.

DETAILED DESCRIPTION

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the content clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention pertains. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice for testing of the invention(s), specificexamples of appropriate materials and methods are described herein.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising,” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

Thiamine pyrophosphate (TPP)-dependent enzymes, including 2-keto aciddecarboxylase and acetolactate synthase, perform a diverse range ofreactions. Acetolactate synthase catalyzes the condensation of twopyruvates to form 2-acetolactate. Isobutanol production from Escherichiacoli has been demonstrated using a 2-keto acid-based pathway. Thispathway contained two TPP-dependent enzymes, acetolactate synthase (ALS)and 2-keto acid decarboxylase (KDC). The present disclosure provides anALS from Bacillus subtilis that catalyzes a decarboxylation of2-ketoisovalerate like KDC both in vivo and in vitro. The disclosurefurther provides engineered metabolic pathways comprising such apolypeptide. The disclosure further provides expression of ALS in E.coli to produce isobutanol without the presence of KDC. Mutationalstudies of ALS have revealed that replacement of Q487 with alaninediminishes only decarboxylase activity and maintains synthase activity.The fact that acetolactate synthase catalyzes a decarboxylation reactionsupports the hypothesis that TPP-dependent enzymes have diverged from acommon ancestor during their evolution.

TPP is a cofactor whose biochemical functions and mechanistic role arewell understood. TPP-dependent enzymes perform a diverse range ofreactions, including non-oxidative decarboxylation of 2-keto acids,oxidative decarboxylation of 2-keto acids, and carboligation. It isgenerally accepted that the initial step in catalysis by these enzymesis the deprotonation of the thiazolium ring at the C-2 atom involving aconserved glutamate, N-1′, and the 4′-amino group. Protein sequencecomparisons of various TPP-dependent enzymes reveal that these enzymesshare high sequence homology with each other. In addition, proteinstructure comparisons of TPP-dependent enzymes: transketolase, pyruvateoxidase, and pyruvate decarboxylase (PDC), indicated that theirTPP-binding sites are very similar.

ALS is part of the valine biosynthesis pathway and catalyzes the aldocondensation of two molecules of pyruvate to 2-acetolactate. The overallreaction catalyzed by ALS is irreversible because of CO₂ evolution. Thefirst step in catalysis is the ionization of the thiazolium ring of TPP.The highly reactive tricyclic intermediate first forms and this reactswith the first pyruvate that then decarboxylates to give the relativelynon-reactive enamine. Because this intermediate is stable, the enzymecan pause midway through the catalytic cycle while releasing CO₂ andadmitting the second molecule of pyruvate. The tricyclic-carbanion thenforms, followed by reacting with the second pyruvate. Deprotonationfollowed by C—C bond breakage completes the reaction, producing2-acetolactate.

KDC is a non-oxidative TPP-dependent enzyme. KDCs are rare in bacteria,being more frequent in plants, yeasts, and fungi. A number of KDCs havebeen identified in various organisms, and these enzymes include PDC,phenylpyruvate decarboxylase, branched-chain 2-keto acid decarboxylase,2-ketoglutarate decarboxylase, and indole-3-pyruvate decarboxylase.2-Keto acids are intermediates in amino acid biosynthesis pathways andcan be converted to aldehydes by KDCs in the Ehrlich pathway.

In a previous study to produce isobutanol, glucose was converted to2-ketoisovalerate through over-expressed AlsS (Bacillus subtilis), IlvC(E. coli), and IlvD (E. coli) (FIG. 1) (Atsumi and Liao, Nature,451:86-89, 2008). The resulting 2-ketoisovalerate (2-KIV) is thenconverted to isobutanol using Kivd (Lactoccus lactis) and Adh2(Saccharomyces cerevisiae) (FIG. 1A). This strain produced 6.8 g/Lisobutanol in 24 hrs and more than 20 g/L in 112 hrs (id.).

The present disclosure describes the use of ALS from B. subtilis (SEQ IDNO:1 and 2) in a recombinant microorganism for the generation ofbiofuels including isobutanol. Furthermore, the disclosure provided avariant ALS that can be used in such microorganisms. Both the wild-typeand variant ALS can be engineered into a micoroorganism to generate arecombinant metabolic pathway for the generation of a biofuel such asisobutanol in the absence of a KDC.

While these polypeptides and variants will be described in more detailbelow, it is understood that polypeptides of the disclosure can containone or more modified amino acids or additional conservative amino acidsubstitutions. The presence of modified amino acids can be advantageousin, for example, (a) increasing polypeptide in vivo half-life, (b)reducing or increasing polypeptide antigenicity, and (c) increasingpolypeptide storage stability. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N—X—S/T motifs during expression ineukaryotic cells) or modified by synthetic means. Accordingly, a“mutant”, “variant” or “modified” protein, enzyme, polynucleotide, gene,or cell, means a protein, enzyme, polynucleotide, gene, or cell, thathas been altered or derived, or is in some way different or changed,from a parent protein, enzyme, polynucleotide, gene, or cell. A mutantor modified protein or enzyme is usually, although not necessarily,expressed from a mutant polynucleotide or gene.

A “parent” protein, enzyme, polynucleotide, gene, or cell, is anyprotein, enzyme, polynucleotide, gene, or cell, from which any otherprotein, enzyme, polynucleotide, gene, or cell, is derived or made,using any methods, tools, or techniques, and whether or not the parentis itself native or mutant. A parent polynucleotide or gene encodes fora parent protein or enzyme.

A “mutation” means any process or mechanism resulting in a mutantprotein, enzyme, polynucleotide, gene, or cell. This includes anymutation in which a protein, enzyme, polynucleotide, or gene sequence isaltered, and any detectable change in a cell arising from such amutation. Typically, a mutation occurs in a polynucleotide or genesequence, by point mutations, deletions, or insertions of single ormultiple nucleotide residues. A mutation includes polynucleotidealterations arising within a protein-encoding region of a gene as wellas alterations in regions outside of a protein-encoding sequence, suchas, but not limited to, regulatory or promoter sequences. A mutation ina gene can be “silent”, i.e., not reflected in an amino acid alterationupon expression, leading to a “sequence-conservative” variant of thegene. This generally arises when one amino acid corresponds to more thanone codon.

Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenylated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a pegylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998), Protein Protocols on CD-ROM (Humana Press, Towata,N.J.).

A “protein” or “polypeptide”, which terms are used interchangeablyherein, comprises one or more chains of chemical building blocks calledamino acids that are linked together by chemical bonds called peptidebonds. An “enzyme” means any substance, composed wholly or largely ofprotein, that catalyzes or promotes, more or less specifically, one ormore chemical or biochemical reactions. A “native” or “wild-type”protein, enzyme, polynucleotide, gene, or cell, means a protein, enzyme,polynucleotide, gene, or cell that occurs in nature.

The polypeptides disclosed herein are useful in the production ofbiofuels including isobutanol from recombinant microorganisms. In oneembodiment, the microorganism is E. coli. In another embodiment, themicroorganism lacks a gene encoding a 2-keto acid decarboxylase. In yetanother embodiment, the microorganism is an E. coli that is engineeredto express a recombinant ALS or variant thereof and lacks one or moregenes encoding enzymes in a competitive metabolic pathway that causes aflux of substrates away from the desired product (e.g., a biofuel suchas isobutanol).

The production of isobutanol and other fusel alcohols by various yeastspecies, including Saccharomyces cerevisiae, is of special interest tothe distillers of alcoholic beverages, for whom fusel alcoholsconstitute often undesirable off-notes. Production of isobutanol inwild-type yeasts has been documented on various growth media, rangingfrom grape must from winemaking (Romano, et al., (2003) World J.Microbiol. Biotechnol. 19:311-315), in which 12-219 mg/L isobutanol wereproduced, to supplemented minimal media (Oliviera, et al. (2005) WorldJ. Microbiol. Biotechnol. 21:1569-1576), producing 16-34 mg/Lisobutanol. Work from Dickinson, et al., (J. Biol. Chem.272:26871-26878, 1997) has identified the enzymatic steps utilized in apathway converting branch-chain amino acids (e.g., valine or leucine) toisobutanol.

The present disclosure provides metabolically engineered microorganismscomprising biochemical pathways for the production of higher alcoholsincluding isobutanol from a suitable substrate. A metabolicallyengineered microorganism of the disclosure comprises one or morerecombinant polynucleotides within the genome of the organism orexternal to the genome within the organism. The microorganism cancomprise a reduction, disruption, or knockout of a gene found in thewild-type organism and/or introduction of a heterologous polynucleotide.

The present disclosure also includes metabolically engineeredbiosynthetic pathways that utilize an organism's native amino acidpathway. Biofuel production utilizing the organism's native amino acidbiosynthetic pathways offers several advantages. Not only does it avoidthe difficulty of expressing a large set of foreign genes but it alsominimizes the possible accumulation of toxic intermediates. Contrary tothe butanol production pathway found in many species of Clostridium, theengineered amino acid biosynthetic routes for biofuel productioncircumvent the need to involve oxygen-sensitive enzymes andCoA-dependent intermediates.

In one embodiment, the disclosure provides a recombinant microorganismcomprising elevated expression of at least one target enzyme as comparedto a parental microorganism or encodes an enzyme not found in theparental organism. In a specific embodiment, the enzyme comprises anacetolactate synthase (ALS). In yet another embodiment, the enzymecomprises an acetolactate synthase from B. subtilis. In a furtherembodiment, the enzyme comprises a sequence as set forth in SEQ ID NO:2or a variant having at least 80%, 90%, 95%, 98%, or 99% identity to SEQID NO:2 and having acetolactate synthase activity. In some embodimentswherein the microorganism comprises an acetolactate synthase from B.subtilis or a derivative thereof, the microorganism lacks a 2-keto aciddecarboxylase. In a further embodiment, the enzyme comprises a sequenceas set forth in SEQ ID NO:7 or a variant having at least 80%, 90%, 95%,98%, or 99% identity to SEQ ID NO:7, and having acetolactate synthaseactivity.

In another or further embodiment, the microorganism comprises areduction, disruption, or knockout of at least one gene encoding anenzyme that competes with a metabolite necessary for the production of adesired higher alcohol product. The recombinant microorganism producesat least one metabolite involved in a biosynthetic pathway for theproduction of isobutanol or other a higher alcohol. In general, therecombinant microorganism comprises at least one recombinant metabolicpathway that comprises a target enzyme and can further include areduction in activity or expression of an enzyme in a competitivebiosynthetic pathway. The pathway acts to modify a substrate ormetabolic intermediate in the production of a biofuel such asisobutanol. The target enzyme is encoded by, and expressed from, apolynucleotide derived from a suitable biological source. In someembodiments, the polynucleotide comprises a gene derived from abacterial or yeast source and recombinantly engineered into themicroorganism of the disclosure.

As used herein, the term “metabolically engineered” or “metabolicengineering” involves rational pathway design and assembly ofbiosynthetic genes, genes associated with operons, and control elementsof such polynucleotides, for the production of a desired metabolite,such as a 2-keto acid or a higher alcohol, in a microorganism.“Metabolically engineered” can further include optimization of metabolicflux by regulation and optimization of transcription, translation,protein stability, and protein functionality using genetic engineeringand appropriate culture conditions including the reduction of,disruption, or knocking out of, a competing metabolic pathway thatcompetes with an intermediate leading to a desired pathway. Abiosynthetic gene can be heterologous to the host microorganism, eitherby virtue of being foreign to the host, or by being modified bymutagenesis, recombination, and/or association with a heterologousexpression control sequence in an endogenous host cell. In oneembodiment, where the polynucleotide is xenogenetic to the hostorganism, the polynucleotide can be codon optimized.

The term “biosynthetic pathway”, also referred to as “metabolicpathway”, refers to a set of anabolic or catabolic biochemical reactionsfor converting (transmuting) one chemical species into another. Geneproducts belong to the same “metabolic pathway” if they, in parallel orin series, act on the same substrate, produce the same product, or acton or produce a metabolic intermediate (i.e., metabolite) between thesame substrate and metabolite end product.

For example, L-valine is synthesized through a biosynthetic pathwayinherent to L-valine which diverges from the intermediate(2-ketoisovalerate) of the L-leucine biosynthesis system. InEscherichia, the biosynthesis of L-valine and biosynthesis inherent toL-leucine are carried out by a group of enzymes encoded by ilvGMEDAoperon and those encoded by leuABCD operon, respectively.

The ilvGMEDA operon includes ilvG, ilvM, ilvE, ilvD, and ilvA genes.Among them, ilvG encodes acetolactate synthase II large subunit, ilvMencodes acetolactate II synthase small subunit, ilvE encodesbranched-chain amino acid amino transferase, ilvD encodes dihydroxyaciddehydratase, and ilvA encodes threonine deaminase. In some species andstrains of E. coli, ilvG and ilvM are silent.

The leuABCD operon includes leuA, leuB, leuC, and leuD genes. Amongthem, leuA encodes α-isopropylmalate synthase, leuB encodesβ-isopropylmalate dehydrogenase, and leuC and leuD encodeα-isopropylmalate isomerase. Of these enzymes, α-isopropylmalatesynthase catalyzes the synthetic reaction from α-ketoisovalerate toα-isopropylmalate, α-isopropylmalate isomerase catalyzes theisomerization reaction from α-isopropylmalate to β-isopropylmalate, andβ-isopropylmalate dehydrogenase catalyzes the dehydrogenation reactionfrom β-isopropylmalate to α-ketoisocaproic acid, which is the finalintermediate of L-leucine biosynthesis. Escherichia possess four kindsof transaminases, namely, transaminase A (aspartate-glutamateaminotransferase) encoded by aspC gene, transaminase B (BCAAaminotransferase) encoded by ilvE gene which is included in ilvGMEDAoperon, transaminase C (alanine-valine aminotransferase) encoded by avtAgene and transaminase D (tyrosine aminotransferase) encoded by tyrBgene. These enzymes participate in various amination reactions. Of theseenzymes, transaminase B and transaminase D catalyze the above-mentionedamination reaction from α-ketoisocaproic acid to L-leucine. TransaminaseC and transaminase D catalyze the final step of L-valine biosyntheticpathway, which includes a common pathway among the L-valine biosynthesisand L-leucine biosynthesis pathways.

Expression of ilvBN gene encoding acetohydroxy acid synthase I suffersconcerted repression by L-valine and L-leucine, expression of ilvGM geneencoding acetohydroxy acid synthase II suffers concerted repression byL-isoleucine, L-valine, and L-leucine, and expression of ilvIH geneencoding acetohydroxy acid synthase III suffers repression by L-leucine.

The term “substrate” or “suitable substrate” refers to any substance orcompound that is converted or meant to be converted into anothercompound by the action of an enzyme. The term includes not only a singlecompound, but also combinations of compounds, such as solutions,mixtures, and other materials which contain at least one substrate, orderivatives thereof. Further, the term “substrate” encompasses not onlycompounds that provide a carbon source suitable for use as a startingmaterial, such as any biomass derived sugar, but also intermediate andend product metabolites used in a pathway associated with ametabolically engineered microorganism as described herein. A “biomassderived sugar” includes, but is not limited to, molecules such asglucose, sucrose, mannose, xylose, and arabinose. The term biomassderived sugar encompasses suitable carbon substrates ordinarily used bymicroorganisms, such as 6 carbon sugars, including but not limited toglucose, lactose, sorbose, fructose, idose, galactose, and mannose, allin either D or L form, or a combination of 6 carbon sugars, such asglucose and fructose, and/or 6 carbon sugar acids including, but notlimited to, 2-keto-L-gulonic acid, idonic acid (IA), gluconic acid (GA),6-phosphogluconate, 2-keto-D-gluconic acid (2 KDG), 5-keto-D-gluconicacid, 2-ketogluconatephosphate, 2,5-diketo-L-gulonic acid,2,3-L-diketogulonic acid, dehydroascorbic acid, erythorbic acid (EA),and D-mannonic acid.

The term “alcohol” includes, for example, 1-propanol, isobutanol,1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, or 2-phenylethanol.The term “1-butanol” or “n-butanol” generally refers to a straight chainisomer with the alcohol functional group at the terminal carbon. Thestraight chain isomer with the alcohol at an internal carbon issec-butanol or 2-butanol. The branched isomer with the alcohol at aterminal carbon is isobutanol, and the branched isomer with the alcoholat the internal carbon is tert-butanol.

Recombinant microorganisms provided herein can express a plurality oftarget enzymes involved in pathways for the production of, for example,isobutanol, 1-propanol, 1-butanol, 2-methyl-1-butanol,3-methyl-1-butanol, or 2-phenylethanol using a suitable carbonsubstrate.

Accordingly, metabolically “engineered” or “modified” microorganisms areproduced via the introduction of genetic material into a host orparental microorganism of choice, thereby modifying or altering thecellular physiology and biochemistry of the microorganism. Through theintroduction of genetic material, the parental microorganism acquiresnew properties, e.g., the ability to produce new, or greater quantitiesof, an intracellular metabolite. In an illustrative embodiment, theintroduction of genetic material into a parental microorganism resultsin a new or modified ability to produce an alcohol such as isobutanol,1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, or2-phenylethanol. The genetic material introduced into the parentalmicroorganism contains gene(s), or parts of genes, coding for one ormore of the enzymes involved in a biosynthetic pathway for theproduction of an alcohol and may also include additional elements forthe expression and/or regulation of expression of these genes, e.g.,promoter sequences.

An engineered or modified microorganism can also include in thealternative or in addition to the introduction of a genetic materialinto a host or parental micoorganism, the disruption, deletion, orknocking out of a gene or polynucleotide to alter the cellularphysiology and biochemistry of the microorganism. Through the reduction,disruption, or knocking out of a gene or polynucleotide themicroorganism acquires new or improved properties (e.g., the ability toproduce a new or greater quantities of an intracellular metabolite,improve the flux of a metabolite down a desired pathway, and/or reducethe production of undesirable by-products).

The disclosure demonstrates that the expression of one or moreheterologous polynucleotide(s) or over-expression of one or moreheterologous polynucleotide(s) encoding a polypeptide having bothacetolactate synthase and keto acid decarboxylase activity are useful inthe generation of isobutanol. In one specific embodiment, the disclosuredemonstrates that with over-expression of the heterologous alsS genefrom B. subtilis and adh2, and the ilvC and IlvD from E. coli, theproduction of isobutanol can be obtained.

Microorganisms provided herein are modified to produce metabolites inquantities not available in the parental microorganism. A “metabolite”refers to any substance produced by metabolism or a substance necessaryfor or taking part in a particular metabolic process. A metabolite canbe an organic compound that is a starting material (e.g., glucose orpyruvate), an intermediate (e.g., 2-keto acid), or an end product (e.g.,1-propanol, isobutanol, 1-butanol, 2-methyl-1-butanol,3-methyl-1-butanol, or 2-phenylethanol) of metabolism. Metabolites canbe used to construct more complex molecules, or they can be broken downinto simpler ones. Intermediate metabolites may be synthesized fromother metabolites, perhaps used to make more complex substances, orbroken down into simpler compounds, often with the release of chemicalenergy.

Exemplary metabolites include glucose, pyruvate, 1-propanol, isobutanol,1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol,and 2-ketoisovalerate or 2-ketovaleric acids. As depicted in FIG. 1A,exemplary 2-keto acid intermediates include 2-ketobutyrate,2-ketoisovalerate, 2-ketovalerate, 2-keto 3-methylvalerate, 2-keto4-methyl-pentanoate, and phenylpyruvate.

Accordingly, provided herein are recombinant microorganisms that produceisobutanol and in some aspects may include the elevated expression oftarget enzymes such as acetohydroxy acid synthase (e.g., ilvIH operon,or any other als containing operon), acetohydroxy acid isomeroreductase(e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD), and alcoholdehydrogenase (e.g., ADH2). The microorganism can further include thedeletion or inhibition of expression of an ethanol dehydrogenase (e.g.,an adhE), ldh (e.g., an ldhA), frd (e.g., an frdB, an frdC or an frdBC),fnr, leuA, ilvE, poxB, ilvA, pflB, or pta gene, or any combinationthereof, to increase the availability of pyruvate or reduce enzymes thatcompete for a metabolite in a desired biosynthetic pathway. In someaspects the recombinant microorganism can include the elevatedexpression of acetolactate synthase (e.g., alsS), acetohydroxy acidisomeroreductase (e.g., ilvC), dihydroxy-acid dehydratase (e.g., ilvD),and alcohol dehydrogenase (e.g., ADH2). With reference to alcoholdehydrogenases, although ethanol dehydrogenase is an alcoholdehydrogenase, the synthesis of ethanol is undesirable as a by-productin the biosynthetic pathways. Accordingly, reference to an increase inalcohol dehydrogenase activity or expression in a microorganismspecifically excludes ethanol dehydrogenase activity.

As previously noted, the target enzymes described throughout thisdisclosure generally produce metabolites. For example, the enzymesacetolactate synthase (alsS), acetohydroxy acid isomeroreductase (ilvC),and dihydroxy-acid dehydratase (ilvD) can produce 2-ketoisovalerate froma substrate that includes pyruvate. In addition, the target enzymesdescribed throughout this disclosure are encoded by polynucleotides. Forexample, acetohydroxy acid isomeroreductase can be encoded by apolynucleotide derived from an ilvC gene. Dihydroxy-acid dehydratase canbe encoded by a polynucleotide derived from an ilvD gene. Alcoholdehydrogenase can be encoded by a polynucleotide derived from an ADH2gene. Additional enzymes and exemplary genes are described throughoutthis document. Homologs of the various polypeptides and polynucleotidescan be derived from any biologic source that provides a suitablepolynucleotide encoding a suitable enzyme. Homologs, for example, can beidentified by reference to various databases.

The disclosure identifies specific genes useful in the methods,compositions, and organisms of the disclosure; however it will berecognized that absolute identity to such genes is not necessary. Forexample, changes in a particular gene or polynucleotide comprising asequence encoding a polypeptide or enzyme can be performed and screenedfor activity. Typically such changes comprise conservative mutationsand/or silent mutations. Such modified or mutated polynucleotides andpolypeptides can be screened for expression of a functional enzymeactivity using methods known in the art.

Due to the inherent degeneracy of the genetic code, otherpolynucleotides which encode substantially the same or a functionallyequivalent polypeptide can also be used to clone and express thepolynucleotides encoding such enzymes.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,typical stop codons for S. cerevisiae and mammals are UAA and UGA,respectively. The typical stop codon for monocotyledonous plants is UGA,whereas insects and E. coli commonly use UAA as the stop codon (Dalphinet al. (1996) Nucl. Acids Res. 24:216-218). Methodology for optimizing anucleotide sequence for expression in a plant is provided, for example,in U.S. Pat. No. 6,015,891, and the references cited therein, allincorporated herein in their entirety.

In addition, homologs of enzymes useful for generating metabolites areencompassed by the microorganisms and methods provided herein. The term“homologs” used with respect to an original enzyme or gene of a firstfamily or species refers to distinct enzymes or genes of a second familyor species which are determined by functional, structural, or genomicanalyses to be an enzyme or gene of the second family or species whichcorresponds to the original enzyme or gene of the first family orspecies. Most often, homologs will have functional, structural, and/orgenomic similarities. Techniques are known to the skilled artisan bywhich homologs of an enzyme or gene can readily be cloned using geneticprobes and PCR. Identity of cloned sequences as a homolog can beconfirmed using functional assays and/or by genomic mapping of thegenes.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.)

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at least30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity. To determine the percent identityof two amino acid sequences, or of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In one embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,typically at least 40%, more typically at least 50%, even more typicallyat least 60%, and even more typically at least 70%, 80%, 90%, or 100% ofthe length of the reference sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. For example, reference to anals gene includes homologs from other organisms encoding an enzymehaving substantially similar enzymatic activity, as well as genes havingat least 30, 40, 50, 60, 70, 80, 85, 90, 95, 98, or 99% identity to thereferenced gene and which encodes an enzyme having substantially similarenzymatic activity as the referenced gene. In particular, the homologswill have the highest level of amino acid sequence identity in theregion of the active site or a co-factor binding site, and any otherregion of the enzyme necessary for its function.

It is also understood that an isolated nucleic acid molecule encoding apolypeptide homologous to the enzymes described herein can be created byintroducing one or more nucleotide substitutions, additions, ordeletions into the nucleotide sequence encoding the particularpolypeptide, such that one or more amino acid substitutions, additions,or deletions are introduced into the encoded protein. Mutations can beintroduced into the polynucleotide by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. In contrast tothose positions where it may be desirable to make non-conservative aminoacid substitutions (see above), in some positions it is preferable tomake conservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). In addition, thefollowing five groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2)Asparagine (N), Glutamine (Q); 3) Arginine (R), Lysine (K); 4)Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V),and 5) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions, and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A typical algorithm used for comparing a molecule sequence to a databasecontaining a large number of sequences from different organisms is thecomputer program BLAST (Altschul, J. Mol. Biol. 215:403-441 (1990);Gish, Nature Genet. 3:266-272 (1993); Madden, Meth. Enzymol. 266:131-141(1996); Altschul, Nucl. Acids Res. 25:3389-3402 (1997); Zhang, GenomeRes. 7:649-656 (1997)), especially blastp or tblastn (Altschul, Nucl.Acids Res. 25:3389-3402 (1997)). Typical parameters for BLASTp are:Expectation value: 10 (default); Filter: seg (default); Cost to open agap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments:100 (default); Word size: 11 (default); No. of descriptions: 100(default); Penalty Matrix: BLOWSUM62.

When searching a database containing sequences from a large number ofdifferent organisms, it is typical to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences. For example,percent sequence identity between amino acid sequences can be determinedusing FASTA with its default parameters (a word size of 2 and the PAM250scoring matrix), as provided in GCG Version 6.1, hereby incorporatedherein by reference.

The disclosure provides accession numbers for various genes, homologsand variants useful in the generation of recombinant microorganismsdescribed herein. It is to be understood that homologs and variantsdescribed herein are exemplary and non-limiting. Additional homologs,variants and sequences are available to those of skill in the art usingvarious databases including, for example, the National Center forBiotechnology Information (NCBI), access to which is available on theWorld-Wide-Web.

Ethanol Dehydrogenase (also referred to as Aldehyde-alcoholDeHydrogenase) is encoded in E. coli by adhE. adhE comprises threeactivities: alcohol dehydrogenase (ADH); acetaldehyde/acetyl-CoAdehydrogenase (ACDH); pyruvate-formate-lyase deactivase (PFLdeactivase); PFL deactivase activity catalyzes the quenching of thepyruvate-formate-lyase catalyst in an iron, NAD, and CoA dependentreaction. A decrease or modification of the expression or activity ofethanol dehydrogenase in a transformed host cell can be useful in thepresently disclosed methods for the production of an alcohol, such asisobutanol, 2-methyl 1-butanol, and/or 2-methyl 1-butanol. Homologs areknown in the art (see, e.g., aldehyde-alcohol dehydrogenase (Polytomellasp. Pringsheim 198.80) gi|40644910|emb|CAD42653.2|(40644910);aldehyde-alcohol dehydrogenase (Clostridium botulinum A str. ATCC 3502)gi|148378348|ref|YP_001252889.1|(148378348); aldehyde-alcoholdehydrogenase (Yersinia pestis CO92)gi|16122410|ref|NP_405723.1|(16122410); aldehyde-alcohol dehydrogenase(Yersinia pseudotuberculosis IP 32953)gi|51596429|ref|YP_070620.1|(51596429); aldehyde-alcohol dehydrogenase(Yersinia pestis CO92) gi|115347889|emb|CAL20810.1|(115347889);aldehyde-alcohol dehydrogenase (Yersinia pseudotuberculosis IP 32953)gi|51589711|emb|CAH21341.1|(51589711); aldehyde-alcohol dehydrogenase(Escherichia coli CFT073)gi|26107972|gb|AAN80172.1|AE016760_31(26107972); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Microtus str. 91001)gi|45441777|ref|NP_993316.1|(45441777); aldehyde-alcohol dehydrogenase(Yersinia pestis biovar Microtus str. 91001)gi|45436639|gb|AAS62193.1|(45436639); aldehyde-alcohol dehydrogenase(Clostridium perfringens ATCC 13124)gi|110798574|ref|YP_697219.1|(110798574); aldehyde-alcohol dehydrogenase(Shewanella oneidensis MR-1) gi|24373696|ref|NP_717739.1|(24373696);aldehyde-alcohol dehydrogenase (Clostridium botulinum A str. ATCC 19397)gi|153932445|ref|YP_001382747.1|(153932445); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Antigua str. E1979001)gi|165991833|gb|EDR44134.1|(165991833); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. Hall)gi|153937530|ref|YP_001386298.1|(153937530); aldehyde-alcoholdehydrogenase (Clostridium perfringens ATCC 13124)gi|110673221|gb|ABG82208.1|(110673221); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. Hall)gi|152933444|gb|ABS38943.1|(152933444); aldehyde-alcohol dehydrogenase(Yersinia pestis biovar Orientalis str. F1991016)gi|165920640|gb|EDR37888.1|(165920640); aldehyde-alcohol dehydrogenase(Yersinia pestis biovar Orientalis str. IP275)gi|165913933|gb|EDR32551.1|(165913933); aldehyde-alcohol dehydrogenase(Yersinia pestis Angola) gi|162419116|ref|YP_001606617.1|(162419116);aldehyde-alcohol dehydrogenase (Clostridium botulinum F str. Langeland)gi|153940830|ref|YP_001389712.1|(153940830); aldehyde-alcoholdehydrogenase (Escherichia coli HS)gi|157160746|ref|YP_001458064.1|(157160746); aldehyde-alcoholdehydrogenase (Escherichia coli E24377A)gi|157155679|ref|YP_001462491.1|(157155679); aldehyde-alcoholdehydrogenase (Yersinia enterocolitica subsp. enterocolitica 8081)gi|123442494|ref|YP_001006472.1|(123442494); aldehyde-alcoholdehydrogenase (Synechococcus sp. JA-3-3Ab)gi|86605191|ref|YP_473954.1|(86605191); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 4b F2365)gi|46907864|ref|YP_014253.1|(46907864); aldehyde-alcohol dehydrogenase(Enterococcus faecalis V583) gi|29375484|ref|NP_814638.1|(29375484);aldehyde-alcohol dehydrogenase (Streptococcus agalactiae 2603V/R)gi|22536238|ref|NP_687089.1|(22536238); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. ATCC 19397)gi|152928489|gb|ABS33989.1|(152928489); aldehyde-alcohol dehydrogenase(Escherichia coli E24377A) gi|157077709|gb|ABV17417.1|(157077709);aldehyde-alcohol dehydrogenase (Escherichia coli HS)gi|157066426|gb|ABV05681.1|(157066426); aldehyde-alcohol dehydrogenase(Clostridium botulinum F str. Langeland)gi|152936726|gb|ABS42224.1|(152936726); aldehyde-alcohol dehydrogenase(Yersinia pestis CA88-4125) gi|149292312|gb|EDM42386.1|(149292312);aldehyde-alcohol dehydrogenase (Yersinia enterocolitica subsp.enterocolitica 8081) gi|122089455|emb|CAL12303.1|(122089455);aldehyde-alcohol dehydrogenase (Chlamydomonas reinhardtii)gi|92084840|emb|CAF04128.1|(92084840); aldehyde-alcohol dehydrogenase(Synechococcus sp. JA-3-3Ab) gi|86553733|gb|ABC98691.1|(86553733);aldehyde-alcohol dehydrogenase (Shewanella oneidensis MR-1)gi|24348056|gb|AAN55183.1|AE015655_9(24348056); aldehyde-alcoholdehydrogenase (Enterococcus faecalis V583)gi|29342944|gb|AAO80708.1|(29342944); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 4b F2365)gi|46881133|gb|AAT04430.1|(46881133); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. ½a F6854)gi|47097587|ref|ZP_00235115.1|(47097587); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 4b H7858)gi|47094265|ref|ZP_00231973.1|(47094265); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 4b H7858)gi|47017355|gb|EAL08180.1|(47017355); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. ½a F6854)gi|47014034|gb|EAL05039.1|(47014034); aldehyde-alcohol dehydrogenase(Streptococcus agalactiae 2603V/R)gi|22533058|gb|AAM98961.1|AE014194_6(22533058)p; aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Antigua str. E1979001)gi|166009278|ref|ZP_02230176.1|(166009278); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Orientalis str. IP275)gi|165938272|ref|ZP_02226831.1|(165938272); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Orientalis str. F1991016)gi|165927374|ref|ZP_02223206.1|(165927374); aldehyde-alcoholdehydrogenase (Yersinia pestis Angola)gi|162351931|gb|ABX85879.1|(162351931); aldehyde-alcohol dehydrogenase(Yersinia pseudotuberculosis IP 31758)gi|153949366|ref|YP_001400938.1|(153949366); aldehyde-alcoholdehydrogenase (Yersinia pseudotuberculosis IP 31758)gi|152960861|gb|ABS48322.1|(152960861); aldehyde-alcohol dehydrogenase(Yersinia pestis CA88-4125) gi|149365899|ref|ZP_01887934.1|(149365899);acetaldehyde dehydrogenase (acetylating) (Escherichia coli CFT073)gi|26247570|ref|NP_753610.1|(26247570); aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase; acetaldehyde dehydrogenase(acetylating) (EC 1.2.1.10) (acdh); pyruvate-formate-lyase deactivase(pfl deactivase)) (Clostridium botulinum A str. ATCC 3502)gi|148287832|emb|CAL81898.1|(148287832); aldehyde-alcohol dehydrogenase(Includes: alcohol dehydrogenase (ADH); acetaldehyde dehydrogenase(acetylating) (ACDH); pyruvate-formate-lyase deactivase (PFLdeactivase)) gi|71152980|sp|P0A9Q7.2|ADHE_ECOLI(71152980);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase andacetaldehyde dehydrogenase, and pyruvate-formate-lyase deactivase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|50121254|ref|YP_050421.1|(50121254); aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase and acetaldehyde dehydrogenase, andpyruvate-formate-lyase deactivase (Erwinia carotovora subsp. atrosepticaSCRI1043) gi|49611780|emb|CAG75229.1|(49611780); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase (ADH); acetaldehydedehydrogenase (acetylating) (ACDH))gi|19858620|sp|P33744.3|ADHE_CLOAB(19858620); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase (ADH); acetaldehydedehydrogenase (acetylating) (ACDH); pyruvate-formate-lyase deactivase(PFL deactivase)) gi|71152683|sp|P0A9Q8.2|ADHE_ECO57(71152683);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase;acetaldehyde dehydrogenase (acetylating); pyruvate-formate-lyasedeactivase (Clostridium difficile 630)gi|126697906|ref|YP_001086803.1|(126697906); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase; acetaldehydedehydrogenase (acetylating); pyruvate-formate-lyase deactivase(Clostridium difficile 630) gi|115249343|emb|CAJ67156.1|(115249343);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase (ADH)and acetaldehyde dehydrogenase (acetylating) (ACDH);pyruvate-formate-lyase deactivase (PFL deactivase)) (Photorhabdusluminescens subsp. laumondii TTO1)gi|37526388|ref|NP_929732.1|(37526388); aldehyde-alcohol dehydrogenase 2(includes: alcohol dehydrogenase; acetaldehyde dehydrogenase)(Streptococcus pyogenes str. Manfredo)gi|134271169|emb|CAM29381.1|(134271169); aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase(acetylating) (ACDH); pyruvate-formate-lyase deactivase (PFLdeactivase)) (Photorhabdus luminescens subsp. laumondii TTO1)gi|36785819|emb|CAE14870.1|(36785819); aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase and pyruvate-formate-lyase deactivase(Clostridium difficile 630) gi|126700586|ref|YP_001089483.1|(126700586);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase andpyruvate-formate-lyase deactivase (Clostridium difficile 630)gi|115252023|emb|CAJ69859.1|(115252023); aldehyde-alcohol dehydrogenase2 (Streptococcus pyogenes str. Manfredo)gi|139472923|ref|YP_001127638.1|(139472923); aldehyde-alcoholdehydrogenase E (Clostridium perfringens str. 13)gi|18311513|ref|NP_563447.1|(18311513); aldehyde-alcohol dehydrogenase E(Clostridium perfringens str. 13) gi|18146197|dbj|BAB82237.1|(18146197);aldehyde-alcohol dehydrogenase, ADHE1 (Clostridium acetobutylicum ATCC824) gi|15004739|ref|NP_149199.1|(15004739); aldehyde-alcoholdehydrogenase, ADHE1 (Clostridium acetobutylicum ATCC 824)gi|14994351|gb|AAK76781.1|AE001438_34(14994351); aldehyde-alcoholdehydrogenase 2 (includes: alcohol dehydrogenase (ADH);acetaldehyde/acetyl-CoA dehydrogenase (ACDH))gi|2492737|sp|Q24803.1|ADH2_ENTHI(2492737); alcohol dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16760134|ref|NP_455751.1|(16760134); and alcohol dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi)gi|16502428|emb|CAD08384.1|(16502428)), each sequence associated withthe accession number is incorporated herein by reference in itsentirety.

Lactate Dehydrogenase (also referred to as D-lactate dehydrogenase andfermentive dehydrogenase) is encoded in E. coli by ldhA and catalyzesthe NADH-dependent conversion of pyruvate to D-lactate. A decrease ormodification of the expression or activity of LdhA in a transformed hostcell can be useful in the presently disclosed methods for the productionof an alcohol, such as, for example, isobutanol, 2-methyl 1-butanol, or3-methyl 1-butanol. LdhA homologs and variants are known. In fact thereare currently 1664 bacterial lactate dehydrogenases available throughNCBI. Homologs and variants include, for example, D-lactatedehydrogenase (D-LDH) (fermentative lactate dehydrogenase)gi|1730102|sp|P52643.1|LDHD_ECOLI(1730102); D-lactate dehydrogenasegi|1049265|gb|AAB51772.1|(1049265); D-lactate dehydrogenase (Escherichiacoli APEC O1) gi|117623655|ref|YP_852568.1|(117623655); D-lactatedehydrogenase (Escherichia coli CFT073)gi|26247689|ref|NP_753729.1|(26247689); D-lactate dehydrogenase(Escherichia coli O157:H7 EDL933)gi|15801748|ref|NP_287766.1|(15801748); D-lactate dehydrogenase(Escherichia coli APEC O1) gi|115512779|gb|ABJ00854.1|(115512779);D-lactate dehydrogenase (Escherichia coli CFT073)gi|26108091|gb|AAN80291.1|AE016760_150 (26108091); fermentativeD-lactate dehydrogenase, NAD-dependent (Escherichia coli K12)gi|16129341|ref|NP_415898.1|(16129341); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli UTI89)gi|91210646|ref|YP_540632.1|(91210646); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli K12)gi|1787645|gb|AAC74462.1|(1787645); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli W3110)gi|89108227|ref|AP_002007.1|(89108227); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli W3110)gi|1742259|dbj|BAA14990.1|(1742259); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli UTI89)gi|91072220|gb|ABE07101.1|(91072220); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli O157:H7 EDL933)gi|12515320|gb|AAG56380.1|AE005366_6(12515320); fermentative D-lactatedehydrogenase (Escherichia coli O157:H7 str. Sakai)gi|13361468|dbj|BAB35425.1| (13361468); COG1052: Lactate dehydrogenaseand related dehydrogenases (Escherichia coli 101-1)gi|83588593|ref|ZP_00927217.1|(83588593); COG1052: Lactate dehydrogenaseand related dehydrogenases (Escherichia coli 53638)gi|75515985|ref|ZP_00738103.1| (75515985); COG1052: lactatedehydrogenase and related dehydrogenases (Escherichia coli E22)gi|75260157|ref|ZP_00731425.1|(75260157); COG1052: lactate dehydrogenaseand related dehydrogenases (Escherichia coli F11)gi|75242656|ref|ZP_00726400.1|(75242656); COG1052: lactate dehydrogenaseand related dehydrogenases (Escherichia coli E110019)gi|75237491|ref|ZP_00721524.1|(75237491); COG1052: lactate dehydrogenaseand related dehydrogenases (Escherichia coli B7A)gi|75231601|ref|ZP_00717959.1|(75231601); and COG1052: lactatedehydrogenase and related dehydrogenases (Escherichia coli B171)gi|75211308|ref|ZP_00711407.1|(75211308), each sequence associated withthe accession number is incorporated herein by reference in itsentirety.

Two membrane-bound, FAD-containing enzymes are responsible for thecatalysis of fumarate and succinate interconversion; the fumaratereductase is used in anaerobic growth, and the succinate dehydrogenaseis used in aerobic growth. Fumarate reductase comprises multiplesubunits (e.g., frdA, B, and C in E. coli). Modification of any one ofthe subunits can result in the desired activity herein. For example, aknockout of frdB, frdC or frdBC is useful in the methods of thedisclosure. Frd homologs and variants are known. Homologs and variantsincludes, for example, fumarate reductase subunit D (fumarate reductase13 kDa hydrophobic protein) gi|67463543|sp|P0A8Q3.1|FRDD_ECOLI(67463543); fumarate reductase subunit C (fumarate reductase 15 kDahydrophobic protein) gi|1346037|sp|P20923.2|FRDC_PROVU(1346037);fumarate reductase subunit D (fumarate reductase 13 kDa hydrophobicprotein) gi|120499|sp|P20924.1|FRDD_PROVU(120499); fumarate reductasesubunit C (fumarate reductase 15 kDa hydrophobic protein)gi|67463538|sp|P0A8Q0.1|FRDC_ECOLI(67463538); fumarate reductaseiron-sulfur subunit (Escherichia coli) gi|145264|gb|AAA23438.1|(145264);fumarate reductase flavoprotein subunit (Escherichia coli)gi|145263|gb|AAA23437.1|(145263); fumarate reductase flavoproteinsubunit gi|37538290|sp|P17412.3|FRDA_WOLSU(37538290); fumarate reductaseflavoprotein subunit gi|120489|sp|P00363.3|FRDA_ECOLI(120489); fumaratereductase flavoprotein subunit gi|120490|sp|P20922.1|FRDA_PROVU(120490);Fumarate reductase flavoprotein subunit precursor (flavocytochrome c)(flavocytochrome c3) (Fcc3)gi|119370087|sp|Q07WU7.2|FRDA_SHEFN(119370087); fumarate reductaseiron-sulfur subunit gi|81175308|sp|P0AC47.2|FRDB_ECOLI(81175308);fumarate reductase flavoprotein subunit (flavocytochrome c)(flavocytochrome c3) (Fcc3)gi|119370088|sp|P0C278.1|FRDA_SHEFR(119370088); frd operonuncharacterized protein C gi|140663|sp|P20927.1|YFRC_PROVU(140663); frdoperon probable iron-sulfur subunit Agi|140661|sp|P20925.1|YFRA_PROVU(140661); fumarate reductase iron-sulfursubunit gi|120493|sp|P20921.2|FRDB_PROVU(120493); fumarate reductaseflavoprotein subunit gi|2494617|sp|O06913.2|FRDA_HELPY(2494617);fumarate reductase flavoprotein subunit precursor (Iron(III)-inducedflavocytochrome C3) (Ifc3) gi|13878499|sp|Q9Z4P0.1|FRD2_SHEFN(13878499);fumarate reductase flavoprotein subunitgi|54041009|sp|P64174.1|FRDA_MYCTU(54041009); fumarate reductaseflavoprotein subunit gi|54037132|sp|P64175.1|FRDA_MYCBO(54037132);fumarate reductase flavoprotein subunitgi|12230114|sp|Q9ZMP0.1|FRDA_HELPJ(12230114); fumarate reductaseflavoprotein subunit gi|1169737|sp|P44894.1|FRDA_HAEIN(1169737);fumarate reductase flavoprotein subunit (Wolinella succinogenes)gi|13160058|emb|CAA04214.2|(13160058); fumarate reductase flavoproteinsubunit precursor (favocytochrome c) (FL cyt)gi|25452947|sp|P83223.2|FRDA_SHEON (25452947); fumarate reductaseiron-sulfur subunit (Wolinella succinogenes)gi|2282000|emb|CAA04215.1|(2282000); and fumarate reductase cytochrome bsubunit (Wolinella succinogenes) gi|2281998|emb|CAA04213.1|(2281998),each sequence associated with the accession number is incorporatedherein by reference in its entirety.

Acetate kinase is encoded in E. coli by ackA. AckA is involved inconversion of acetyl-coA to acetate. Specifically, ackA catalyzes theconversion of acetyl-phosphate to acetate. A decrease or modification ofthe expression or activity of AckA in a transformed host cell can beuseful in the presently disclosed methods for the production ofisobutanol. AckA homologs and variants are known. The NCBI database listincludes approximately 1450 polypeptides as bacterial acetate kinases.For example, such homologs and variants include acetate kinase(Streptomyces coelicolor A3(2)) gi|21223784|ref|NP_629563.1|(21223784);acetate kinase (Streptomyces coelicolor A3(2))gi|6808417|emb|CAB70654.1|(6808417); acetate kinase (Streptococcuspyogenes M1 GAS) gi|15674332|ref|NP_268506.1|(15674332); acetate kinase(Campylobacter jejuni subsp. jejuni NCTC 11168)gi|15792038|ref|NP_281861.1|(15792038); acetate kinase (Streptococcuspyogenes M1 GAS) gi|13621416|gb|AAK33227.1|(13621416); acetate kinase(Rhodopirellula baltica SH 1) gi|32476009|ref|NP_869003.1|(32476009);acetate kinase (Rhodopirellula baltica SH 1)gi|32472045|ref|NP_865039.1|(32472045); acetate kinase (Campylobacterjejuni subsp. jejuni NCTC 11168) gi|112360034|emb|CAL34826.1|(112360034); acetate kinase (Rhodopirellula baltica SH 1)gi|32446553|emb|CAD76388.1| (32446553); acetate kinase (Rhodopirellulabaltica SH 1) gi|32397417|emb|CAD72723.1| (32397417); AckA (Clostridiumkluyveri DSM 555) gi|153954016|ref|YP_001394781.1| (153954016); acetatekinase (Bifidobacterium longum NCC2705)gi|23465540|ref|NP_696143.1|(23465540); AckA (Clostridium kluyveri DSM555) gi|146346897|gb|EDK33433.1|(146346897); acetate kinase(Corynebacterium diphtheriae) gi|38200875|emb|CAE50580.1|(38200875);acetate kinase (Bifidobacterium longum NCC2705)gi|23326203|gb|AAN24779.1|(23326203); acetate kinase (Acetokinase)gi|67462089|sp|P0A6A3.1| ACKA_ECOLI(67462089); and AckA (Bacilluslicheniformis DSM 13) gi|52349315|gb|AAU41949.1|(52349315), thesequences associated with such accession numbers are incorporated hereinby reference.

Phosphate acetyltransferase (PTA) is encoded in E. coli by the genedesignated pta. PTA is involved in conversion of acetate to acetyl-CoA.Specifically, PTA catalyzes the conversion of acetyl-coA toacetyl-phosphate. A decrease or modification of the expression oractivity of PTA in a transformed host cell can be useful in thepresently disclosed methods for the production of isobutanol. PTAhomologs and variants are known. There are approximately 1075 bacterialphosphate acetyltransferases available on NCBI. For example, suchhomologs and variants include phosphate acetyltransferase Pta(Rickettsia felis URRWXCal2) gi|67004021|gb|AAY60947.1|(67004021);phosphate acetyltransferase (Buchnera aphidicola str. Cc (Cinara cedri))gi|116256910|gb|ABJ90592.1|(116256910); pta (Buchnera aphidicola str. Cc(Cinara cedri)) gi|116515056|ref|YP_802685.1|(116515056); pta(Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis)gi|25166135|dbj|BAC24326.1| (25166135); Pta (Pasteurella multocidasubsp. multocida str. Pm70) gi|12720993|gb|AAK02789.1|(12720993); Pta(Rhodospirillum rubrum) gi|25989720|gb|AAN75024.1| (25989720); pta(Listeria welshimeri serovar 6b str. SLCC5334)gi|116742418|emb|CAK21542.1|(116742418); Pta (Mycobacterium avium subsp.paratuberculosis K-10) gi|41398816|gb|AAS06435.1|(41398816); phosphateacetyltransferase (pta) (Borrelia burgdorferi B31)gi|15594934|ref|NP_212723.1|(15594934); phosphate acetyltransferase(pta) (Borrelia burgdorferi B31) gi|2688508|gb|AAB91518.1|(2688508);phosphate acetyltransferase (pta) (Haemophilus influenzae Rd KW20)gi|1574131|gb|AAC22857.1|(1574131); phosphate acetyltransferase (Pta)(Rickettsia bellii RML369-C) gi|91206026|ref|YP_538381.1|(91206026);phosphate acetyltransferase (Pta) (Rickettsia bellii RML369-C)gi|91206025|ref|YP_538380.1|(91206025); phosphate acetyltransferase(pta) (Mycobacterium tuberculosis F11)gi|148720131|gb|ABR04756.1|(148720131); phosphate acetyltransferase(pta) (Mycobacterium tuberculosis str. Haarlem)gi|134148886|gb|EBA40931.1|(134148886); phosphate acetyltransferase pta(Mycobacterium tuberculosis C) gi|124599819|gb|EAY58829.1|(124599819);phosphate acetyltransferase (Pta) (Rickettsia bellii RML369-C)gi|91069570|gb|ABE05292.1|(91069570); phosphate acetyltransferase Pta(Rickettsia bellii RML369-C) gi|91069569|gb|ABE05291.1|(91069569);phosphate acetyltransferase (pta) (Treponema pallidum subsp. pallidumstr. Nichols) gi|15639088|ref|NP_218534.1|(15639088); and phosphateacetyltransferase (pta) (Treponema pallidum subsp. pallidum str.Nichols) gi|3322356|gb|AAC65090.1|(3322356), each sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

Pyruvate-formate lyase (formate acetyltransferase) is an enzyme thatcatalyzes the conversion of pyruvate to acetyl-coA and formate. It isinduced by pfl-activating enzyme under anaerobic conditions bygeneration of an organic free radical and decreases significantly duringphosphate limitation. A decrease or modification of the expression oractivity of formate acetyltransferase in a transformed host cell can beuseful in the presently disclosed methods for the production ofisobutanol. Formate acetyltransferase is encoded in E. coli by pflB.PFLB homologs and variants are known. Such homologs and variantsinclude, for example, formate acetyltransferase 1 (Pyruvateformate-lyase 1) gi|129879|sp|P09373.2|PFLB_ECOLI(129879); formateacetyltransferase 1 (Yersinia pestis CO92)gi|16121663|ref|NP_404976.1|(16121663); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51595748|ref|YP_069939.1|(51595748); formate acetyltransferase 1(Yersinia pestis biovar Microtus str. 91001)gi|45441037|ref|NP_992576.1|(45441037); formate acetyltransferase 1(Yersinia pestis CO92) gi|115347142|emb|CAL20035.1|(115347142); formateacetyltransferase 1 (Yersinia pestis biovar Microtus str. 91001)gi|45435896|gb|AAS61453.1|(45435896); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51589030|emb|CAH20648.1|(51589030); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16759843|ref|NP_455460.1|(16759843); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56413977|ref|YP_151052.1|(56413977); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi)gi|16502136|emb|CAD05373.1|(16502136); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56128234|gb|AAV77740.1|(56128234); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|82777577|ref|YP_403926.1|(82777577);formate acetyltransferase 1 (Shigella flexneri 2a str. 2457T)gi|30062438|ref|NP_836609.1| (30062438); formate acetyltransferase 1(Shigella flexneri 2a str. 2457T) gi|30040684|gb|AAP16415.1|(30040684);formate acetyltransferase 1 (Shigella flexneri 5 str. 8401)gi|110614459|gb|ABF03126.1|(110614459); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|81241725|gb|ABB62435.1|(81241725);formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933)gi|12514066|gb|AAG55388.1|AE005279_8(12514066); formateacetyltransferase 1 (Yersinia pestis KIM)gi|22126668|ref|NP_670091.1|(22126668); formate acetyltransferase 1(Streptococcus agalactiae A909) gi|76787667|ref|YP_330335.1|(76787667);formate acetyltransferase 1 (Yersinia pestis KIM)gi|21959683|gb|AAM86342.1|AE013882_3(21959683); formateacetyltransferase 1 (Streptococcus agalactiae A909)gi|76562724|gb|ABA45308.1|(76562724); formate acetyltransferase 1(Yersinia enterocolitica subsp. enterocolitica 8081)gi|123441844|ref|YP_001005827.1|(123441844); formate acetyltransferase 1(Shigella flexneri 5 str. 8401)gi|110804911|ref|YP_688431.1|(110804911); formate acetyltransferase 1(Escherichia coli UTI89) gi|91210004|ref|YP_539990.1|(91210004); formateacetyltransferase 1 (Shigella boydii Sb227)gi|82544641|ref|YP_408588.1|(82544641); formate acetyltransferase 1(Shigella sonnei Ss046) gi|74311459|ref|YP_309878.1|(74311459); formateacetyltransferase 1 (Klebsiella pneumoniae subsp. pneumoniae MGH 78578)gi|152969488|ref|YP_001334597.1|(152969488); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi Ty2)gi|29142384|ref|NP_805726.1|(29142384) formate acetyltransferase 1(Shigella flexneri 2a str. 301) gi|24112311|ref|NP_706821.1| (24112311);formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933)gi|158007641 ref|NP_286778.1|(15800764); formate acetyltransferase 1(Klebsiella pneumoniae subsp. pneumoniae MGH 78578)gi|150954337|gb|ABR76367.1|(150954337); formate acetyltransferase 1(Yersinia pestis CA88-4125) gi|149366640|ref|ZP_01888674.1| (149366640);formate acetyltransferase 1 (Yersinia pestis CA88-4125)gi|149291014|gb|EDM41089.1|(149291014); formate acetyltransferase 1(Yersinia enterocolitica subsp. enterocolitica 8081)gi|122088805|emb|CAL11611.1|(122088805); formate acetyltransferase 1(Shigella sonnei Ss046) gi|73854936|gb|AAZ87643.1|(73854936); formateacetyltransferase 1 (Escherichia coli UTI89) gi|91071578|gb|ABE06459.1|(91071578); formate acetyltransferase 1 (Salmonella enterica subsp.enterica serovar Typhi Ty2) gi|29138014|gb|AAO69575.1|(29138014);formate acetyltransferase 1 (Shigella boydii Sb227)gi|81246052|gb|ABB66760.1|(81246052); formate acetyltransferase 1(Shigella flexneri 2a str. 301) gi|24051169|gb|AAN42528.1|(24051169);formate acetyltransferase 1 (Escherichia coli O157:H7 str. Sakai)gi|13360445|dbj|BAB34409.1|(13360445); formate acetyltransferase 1(Escherichia coli O157:H7 str. Sakai)gi|15830240|ref|NP_309013.1|(15830240); formate acetyltransferase I(pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|36784986|emb|CAE13906.1|(36784986); formate acetyltransferase I(pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|37525558|ref|NP_928902.1|(37525558); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu50)gi|14245993|dbj|BAB56388.1|(14245993); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu50)gi|15923216|ref|NP_370750.1|(15923216); formate acetyltransferase(pyruvate formate-lyase) gi|81706366|sp|Q7A7X6.1|PFLB_STAAN(81706366);formate acetyltransferase (pyruvate formate-lyase)gi|81782287|sp|Q99WZ7.1|PFLB_STAAM(81782287); formate acetyltransferase(pyruvate formate-lyase) gi|81704726|sp|Q7A1W9.1|PFLB_STAAW(81704726);formate acetyltransferase (Staphylococcus aureus subsp. aureus Mu3)gi|156720691|dbj|BAF77108.1| (156720691); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|50121521|ref|YP_050688.1|(50121521); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCRI1043)gi|49612047|emb|CAG75496.1|(49612047); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|150373174|dbj|BAF66434.1|(150373174); formate acetyltransferase(Shewanella oneidensis MR-1) gi|24374439|ref|NP_718482.1|(24374439);formate acetyltransferase (Shewanella oneidensis MR-1)gi|24349015|gb|AAN55926.1|AE015730_3(24349015); formateacetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165976461|ref|YP_001652054.1| (165976461); formate acetyltransferase(Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165876562|gb|ABY69610.1|(165876562); formate acetyltransferase(Staphylococcus aureus subsp. aureus MW2)gi|21203365|dbj|BAB94066.1|(21203365); formate acetyltransferase(Staphylococcus aureus subsp. aureus N315)gi|13700141|dbj|BAB41440.1|(13700141); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|151220374|ref|YP_001331197.1|(151220374); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu3)gi|156978556|ref|YP_001440815.1| (156978556); formate acetyltransferase(Synechococcus sp. JA-2-3B′a(2-13))gi|86607744|ref|YP_476506.1|(86607744); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86605195|ref|YP_473958.1|(86605195);formate acetyltransferase (Streptococcus pneumoniae D39)gi|116517188|ref|YP_815928.1|(116517188); formate acetyltransferase(Synechococcus sp. JA-2-3B′a(2-13))gi|86556286|gb|ABD01243.1|(86556286); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86553737|gb|ABC98695.1|(86553737);formate acetyltransferase (Clostridium novyi NT)gi|118134908|gb|ABK61952.1| (118134908); formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49482458|ref|YP_039682.1|(49482458); and formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49240587|emb|CAG39244.1|(49240587), each sequence associated with theaccession number is incorporated herein by reference in its entirety.

Alpha isopropylmalate synthase (EC 2.3.3.13, sometimes referred to a2-isopropylmalate synthase, alpha-IPM synthetase) catalyzes thecondensation of the acetyl group of acetyl-CoA with3-methyl-2-oxobutanoate (2-oxoisovalerate) to form3-carboxy-3-hydroxy-4-methylpentanoate (2-isopropylmalate). Alphaisopropylmalate synthase is encoded in E. coli by leuA. LeuA homologsand variants are known. Such homologs and variants include, for example,2-isopropylmalate synthase (Corynebacterium glutamicum)gi|452382|emb|CAA50295.1|(452382); 2-isopropylmalate synthase(Escherichia coli K12) gi|16128068|ref|NP_414616.1|(16128068);2-isopropylmalate synthase (Escherichia coli K12)gi|1786261|gb|AAC73185.1|(1786261); 2-isopropylmalate synthase(Arabidopsis thaliana) gi|15237194|ref|NP_197692.1|(15237194);2-isopropylmalate synthase (Arabidopsis thaliana)gi|42562149|ref|NP_173285.2|(42562149); 2-isopropylmalate synthase(Arabidopsis thaliana) gi|15221125|ref|NP_177544.1| (15221125);2-isopropylmalate synthase (Streptomyces coelicolor A3(2))gi|32141173|ref|NP_733575.1|(32141173); 2-isopropylmalate synthase(Rhodopirellula baltica SH 1) gi|32477692|ref|NP_870686.1|(32477692);2-isopropylmalate synthase (Rhodopirellula baltica SH 1)gi|32448246|emb|CAD77763.1|(32448246); 2-isopropylmalate synthase(Akkermansia muciniphila ATCC BAA-835)gi|166241432|gb|EDR53404.1|(166241432); 2-isopropylmalate synthase(Herpetosiphon aurantiacus ATCC 23779)gi|159900959|ref|YP_001547206.1|(159900959); 2-isopropylmalate synthase(Dinoroseobacter shibae DFL 12)gi|159043149|ref|YP_001531943.1|(159043149); 2-isopropylmalate synthase(Salinispora arenicola CNS-205)gi|159035933|ref|YP_001535186.1|(159035933); 2-isopropylmalate synthase(Clavibacter michiganensis subsp. michiganensis NCPPB 382)gi|148272757|ref|YP_001222318.1|(148272757); 2-isopropylmalate synthase(Escherichia coli B) gi|124530643|ref|ZP_01701227.1|(124530643);2-isopropylmalate synthase (Escherichia coli C str. ATCC 8739)gi|124499067|gb|EAY46563.1|(124499067); 2-isopropylmalate synthase(Bordetella pertussis Tohama I) gi|33591386|ref|NP_879030.1|(33591386);2-isopropylmalate synthase (Polynucleobacter necessarius STIR1)gi|164564063|ref|ZP_02209880.1|(164564063); 2-isopropylmalate synthase(Polynucleobacter necessarius STIR1)gi|164506789|gb|EDQ94990.1|(164506789); and 2-isopropylmalate synthase(Bacillus weihenstephanensis KBAB4)gi|163939313|ref|YP_001644197.1|(163939313), any sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

BCAA aminotransferases catalyze the formation of branched chain aminoacids (BCAA). A number of such aminotransferases are known and areexemplified by ilvE in E. coli. A reduction in the expression oractivity or some aminotransferases in a host cell, such as ilvE, can beadvantageous in the methods of the present disclosure. Exemplaryhomologs and variants include sequences designated by the followingaccession numbers: ilvE (Microcystis aeruginosa PCC 7806)gi|159026756|emb|CAO86637.1|(159026756); IlvE (Escherichia coli)gi|87117962|gb|ABD20288.1|(87117962); IlvE (Escherichia coli)gi|87117960|gb|ABD20287.1|(87117960); IlvE (Escherichia coli)gi|87117958|gb|ABD20286.1|(87117958); IlvE (Shigella flexneri)gi|87117956|gb|ABD20285.1|(87117956); IlvE (Shigella flexneri)gi|87117954|gb|ABD20284.1|(87117954); IlvE (Shigella flexneri)gi|87117952|gb|ABD20283.1|(87117952); IlvE (Shigella flexneri)gi|87117950|gb|ABD20282.1|(87117950); IlvE (Shigella flexneri)gi|87117948|gb|ABD20281.1|(87117948); IlvE (Shigella flexneri)gi|87117946|gb|ABD20280.1|(87117946); IlvE (Shigella flexneri)gi|87117944|gb|ABD20279.1|(87117944); IlvE (Shigella flexneri)gi|87117942|gb|ABD20278.1|(87117942); IlvE (Shigella flexneri)gi|87117940|gb|ABD20277.1|(87117940); IlvE (Shigella flexneri)gi|87117938|gb|ABD20276.1|(87117938); IlvE (Shigella dysenteriae)gi|87117936|gb|ABD20275.1|(87117936); IlvE (Shigella dysenteriae)gi|87117934|gb|ABD20274.1|(87117934); IlvE (Shigella dysenteriae)gi|87117932|gb|ABD20273.1| (87117932); IlvE (Shigella dysenteriae)gi|87117930|gb|ABD20272.1|(87117930); and IlvE (Shigella dysenteriae)gi|87117928|gb|ABD20271.1|(87117928), each sequence associated with theaccession number is incorporated herein by reference.

Tyrosine aminotransferases catalyzes transamination for bothdicarboxylic and aromatic amino-acid substrates. A tyrosineaminotransferase of E. coli is encoded by the gene tyrB. A reduction inthe expression or activity or some aminotransferases in a host cell,such as TyrB, can be advantageous in the methods of the presentdisclosure. TyrB homologs and variants are known. For example, suchhomologs and variants include tyrB (Bordetella petrii)gi|163857093|ref|YP_001631391.11 (163857093); tyrB (Bordetella petrii)gi|16326082|emb|CAP43123.1|(163260821); aminotransferasegi|551844|gb|AAA24704.1| (551844); aminotransferase (Bradyrhizobium sp.BTAil) gi|146404387|gb|ABQ32893.1| (146404387); tyrosineaminotransferase TyrB (Salmonella enterica)gi|4775574|emb|CAB40973.2|(4775574); tyrosine aminotransferase(Salmonella typhimurium LT2) gi|16422806|gb|AAL23072.1|(16422806); andtyrosine aminotransferase gi|148085|gb|AAA24703.1|(148085), eachsequence of which is incorporated herein by reference.

Pyruvate oxidase catalyzes the conversion of pyruvate to acetate andCO₂. In E. coli, pyruvate oxidase is encoded by poxB. A reduction in theexpression or activity or some aminotransferases in a host cell, such asPoxB, can be advantageous in the methods of the present disclosure. PoxBand homologs and variants thereof include, for example, pyruvateoxidase; PoxB (Escherichia coli)gi|685128|gb|AAB31180.1∥bbm|348451|bbs|154716(685128); PoxB (Pseudomonasfluorescens) gi|32815820|gb|AAP88293.1| (32815820); poxB (Escherichiacoli) gi|25269169|emb|CAD57486.1|(25269169); pyruvate dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi)gi|16502101|emb|CAD05337.1|(16502101); pyruvate oxidase (Lactobacillusplantarum) gi|41691702|gb|AAS10156.1|(41691702); pyruvate dehydrogenase(Bradyrhizobium japonicum) gi|20257167|gb|AAM12352.1|(20257167);pyruvate dehydrogenase (Yersinia pestis KIM)gi|22126698|ref|NP_670121.1|(22126698); pyruvate dehydrogenase(cytochrome) (Yersinia pestis biovar Antigua str. B42003004)gi|166211240|ref|ZP_02237275.1|(166211240); pyruvate dehydrogenase(cytochrome) (Yersinia pestis biovar Antigua str. B42003004)gi|166207011|gb|EDR51491.1|(166207011); pyruvate dehydrogenase(Pseudomonas syringae pv. tomato str. DC3000)gi|28869703|ref|NP_792322.1|(28869703); pyruvate dehydrogenase(Salmonella typhimurium LT2) gi|16764297|ref|NP_459912.1|(16764297);pyruvate dehydrogenase (Salmonella enterica subsp. enterica serovarTyphi str. CT18) gi|16759808|ref|NP_455425.1|(16759808); pyruvatedehydrogenase (cytochrome) (Coxiella burnetii Dugway 5J108-111)gi|154706110|ref|YP_001424132.1|(154706110); pyruvate dehydrogenase(Clavibacter michiganensis subsp. michiganensis NCPPB 382)gi|148273312|ref|YP_001222873.1|(148273312); pyruvate oxidase(Lactobacillus acidophilus NCFM) gi|58338213|ref|YP_194798.1|(58338213);and pyruvate dehydrogenase (Yersinia pestis CO92)gi|16121638|ref|NP_404951.1|(16121638), the sequences of each accessionnumber are incorporated herein by reference.

L-threonine 3-dehydrogenase (EC 1.1.1.103) catalyzes the conversion ofL-threonine to L-2-amino-3-oxobutanoate. The gene tdh encodes anL-threonine 3-dehydrogenase. There are approximately 700 L-threonine3-dehydrogenases from bacterial organism recognized in NCBI. Varioushomologs and variants of tdh include, for example, L-threonine3-dehydrogenase gi|135560|sp|P07913.1|TDH_ECOLI(135560); L-threonine3-dehydrogenase gi|166227854|sp|A4TSC6.1|TDH_YERPP(166227854);L-threonine 3-dehydrogenasegi|166227853|sp|A1JHX8.1|TDH_YERE8(166227853); L-threonine3-dehydrogenase gi|166227852|sp|A6UBM6.1|TDH_SINMW(166227852);L-threonine 3-dehydrogenasegi|166227851|sp|A1RE07.1|TDH_SHESW(166227851); L-threonine3-dehydrogenase gi|166227850|sp|A0L2Q3.1|TDH_SHESA(166227850);L-threonine 3-dehydrogenasegi|166227849|sp|A4YCC5.1|TDH_SHEPC(166227849); L-threonine3-dehydrogenase gi|166227848|sp|A3QJC8.1|TDH_SHELP(166227848);L-threonine 3-dehydrogenasegi|166227847|sp|A6WUG6.1|TDH_SHEB8(166227847); L-threonine3-dehydrogenase gi|166227846|sp|A3CYN0.1|TDH_SHEB5 (166227846);L-threonine 3-dehydrogenasegi|166227845|sp|A1S1Q3.1|TDH_SHEAM(166227845); L-threonine3-dehydrogenase gi|166227844|sp|A4FND4.1|TDH_SACEN(166227844);L-threonine 3-dehydrogenasegi|166227843|sp|A1SVW5.1|TDH_PSYIN(166227843); L-threonine3-dehydrogenase gi|166227842|sp|A5IGK7.1|TDH_LEGPC(166227842);L-threonine 3-dehydrogenasegi|166227841|sp|A6TFL2.1|TDH_KLEP7(166227841); L-threonine3-dehydrogenase gi|166227840|sp|A4IZ92.1|TDH_FRATW(166227840);L-threonine 3-dehydrogenasegi|166227839|sp|A0Q5K3.1|TDH_FRATN(166227839); L-threonine3-dehydrogenase gi|166227838|sp|A7NDM9.1|TDH_FRATF(166227838);L-threonine 3-dehydrogenasegi|166227837|sp|A7MID0.1|TDH_ENTS8(166227837); and L-threonine3-dehydrogenase gi|166227836|sp|A1AHF3.1|TDH_ECOK1(166227836), thesequences associated with each accession number are incorporated hereinby reference.

Acetohydroxy acid synthases and acetolactate synthases (e.g., alsS)catalyze the synthesis of the branched-chain amino acids (valine,leucine, and isoleucine). IlvH encodes an acetohydroxy acid synthase inE. coli (see, e.g., acetohydroxy acid synthase AHAS III (IlvH)(Escherichia coli) gi|40846|emb|CAA38855.1|(40846), incorporated hereinby reference). Homologs and variants as well as operons comprising ilvHare known and include, for example, ilvH (Microcystis aeruginosa PCC7806) gi|159026908|emb|CAO89159.1|(159026908); IlvH (Bacillusamyloliquefaciens FZB42) gi|154686966|ref|YP_001422127.1|(154686966);IlvH (Bacillus amyloliquefaciens FZB42)gi|154352817|gb|ABS74896.1|(154352817); IlvH (Xenorhabdus nematophila)gi|131054140|gb|ABO32787.1|(131054140); IlvH (Salmonella typhimurium)gi|7631124|gb|AAF65177.1|AF117227_2(7631124), ilvN (Listeria innocua)gi|16414606|emb|CAC97322.1|(16414606); ilvN (Listeria monocytogenes)gi|16411438|emb|CAD00063.1|(16411438); acetohydroxy acid synthase(Caulobacter crescentus) gi|408939|gb|AAA23048.1|(408939); acetohydroxyacid synthase I, small subunit (Salmonella enterica subsp. entericaserovar Typhi) gi|16504830|emb|CAD03199.1| (16504830); acetohydroxy acidsynthase, small subunit (Tropheryma whipplei TW08/27)gi|28572714|ref|NP_789494.1|(28572714); acetohydroxy acid synthase,small subunit (Tropheryma whipplei TWO8/27)gi|28410846|emb|CAD67232.1|(28410846); acetohydroxy acid synthase I,small subunit (Salmonella enterica subsp. enterica serovar Paratyphi Astr. ATCC 9150) gi|56129933|gb|AAV79439.1|(56129933); acetohydroxy acidsynthase small subunit; (Cornybacterium glutamicum)gi|551779|gb|AAA62430.1|(551779); acetohydroxy acid synthase I, smallsubunit (Salmonella enterica subsp. enterica serovar Typhi Ty2)gi|29139650|gb|AAO71216.1|(29139650); acetohydroxy acid synthase smallsubunit (Streptomyces cinnamonensis)gi|5733116|gb|AAD49432.1|AF175526_1|(5733116); and acetohydroxy acidsynthase, large subunit (Cornybacterium glutamicum)gi|400334|gb|AAA62429.1|(400334), the sequences associated with theaccession numbers are incorporated herein by reference.

Acetolactate synthase genes include alsS and ilvI. ALS protein whichhave both acetolactate synthase activity and decarboxylase activity areuseful in the methods of the present disclosure. Homologs of ilvI andalsS are known and can be tested for this dual activity. In addition,the structure of the known proteins can be compared with that of AlsS todetermine homology and that likely possess the dual activity. Knownhomologs can include, for example, acetolactate synthase small subunit(Bifidobacterium longum NCC2705) gi|23325489|gb|AAN24137.1|(23325489);acetolactate synthase small subunit (Geobacillus stearothermophilus)gi|19918933|gb|AAL99357.1|(19918933); acetolactate synthase (Azoarcussp. BH72) gi|119671178|emb|CAL95091.1|(119671178); Acetolactate synthasesmall subunit (Corynebacterium diphtheriae)gi|38199954|emb|CAE49622.1|(38199954); acetolactate synthase (Azoarcussp. BH72) gi|119669739|emb|CAL93652.1|(119669739); acetolactate synthasesmall subunit (Corynebacterium jeikeium K411)gi|68263981|emb|CAI37469.1|(68263981); acetolactate synthase smallsubunit (Bacillus subtilis) gi|1770067|emb|CAA99562.1|(1770067);acetolactate synthase isozyme 1 small subunit (AHAS-I) (acetohydroxyacidsynthase I small subunit) (ALS-I)gi|83309006|sp|P0ADF8.1|ILVN_ECOLI(83309006); acetolactate synthaselarge subunit (Geobacillus stearothermophilus)gi|19918932|gb|AAL99356.1|(19918932); and acetolactate synthase, smallsubunit (Thermoanaerobacter tengcongensis MB4)gi|20806556|ref|NP_621727.1|(20806556), the sequences associated withthe accession numbers are incorporated herein by reference. There areapproximately 1120 ilvB homologs and variants listed in NCBI.

Acetohydroxy acid isomeroreductase is the second enzyme in parallelpathways for the biosynthesis of isoleucine and valine. IlvC encodes anacetohydroxy acid isomeroreductase in E. coli. Homologs and variants ofilvC are known and include, for example, acetohydroxyacidreductoisomerase (Schizosaccharomyces pombe 972 h)gi|162312317|ref|NP_001018845.2|(162312317); acetohydroxyacidreductoisomerase (Schizosaccharomyces pombe)gi|3116142|emb|CAA18891.1|(3116142); acetohydroxyacid reductoisomerase(Saccharomyces cerevisiae YJM789) gi|151940879|gb|EDN59261.1|(151940879); Ilv5p: acetohydroxyacid reductoisomerase (Saccharomycescerevisiae) gi|609403|gb|AAB67753.1|(609403); ACL198Wp (Ashbya gossypiiATCC 10895) gi|45185490|ref|NP_983206.1|(45185490); ACL198Wp (Ashbyagossypii ATCC 10895) gi|44981208|gb|AAS51030.1|(44981208);acetohydroxy-acid isomeroreductase; Ilv5x (Saccharomyces cerevisiae)gi|957238|gb|AAB33579.1∥bbm|369068|bbs|165406(957238); acetohydroxy-acidisomeroreductase; Ilv5 g (Saccharomyces cerevisiae)gi|957236|gb|AAB33578.1∥bbm|369064|bbs|165405(957236); and ketol-acidreductoisomerase (Schizosaccharomyces pombe)gi|2696654|dbj|BAA24000.1|(2696654), each sequence associated with theaccession number is incorporated herein by reference.

Dihydroxy-acid dehydratases catalyze the fourth step in the biosynthesisof isoleucine and valine, the dehydration of 2,3-dihydroxy-isovaleicacid into alpha-ketoisovaleric acid. IlvD and ilv3 encode adihydroxy-acid dehydratase. Homologs and variants of dihydroxy-aciddehydratases are known and include, for example, IlvD (Mycobacteriumleprae) gi|2104594|emb|CAB08798.1|(2104594); dihydroxy-acid dehydratase(Tropheryma whipplei TWO8/27) gi|28410848|emb|CAD67234.1|(28410848);dihydroxy-acid dehydratase (Mycobacterium leprae)gi|13093837|emb|CAC32140.11 (13093837); dihydroxy-acid dehydratase(Rhodopirellula baltica SH 1) gi|32447871|emb|CAD77389.1|(32447871); andputative dihydroxy-acid dehydratase (Staphylococcus aureus subsp. aureusMRSA252) gi|49242408|emb|CAG41121.1|(49242408), each sequence associatedwith the accession numbers are incorporated herein by reference.

2-Keto acid decarboxylases catalyze the conversion of a 2-keto acid tothe respective aldehyde. For example, 2-ketoisovalerate decarboxylasecatalyzes the conversion of 2-ketoisovalerate to isobutyraldehyde. Anumber of 2-keto acid decarboxylases are known and are exemplified bythe pdc, pdc1, pdc5, pdc6, aro10, th13, kdcA and kivd genes. The Alsdescribed herein is intended to replace the 2-keto acid decarboxylaseactivity of this enzyme. In certain examples, a recombinant host celldoes not have to be transformed with a polynucleotide that encodes a2-keto acid decarboxylase reducing the complexity of the recombinantmicroorganism. Exemplary homologs and variants useful for the conversionof a 2-keto acid to the respective aldehyde comprise sequencesdesignated by the following accession numbers and identified enzymaticactivity: gi|44921617|gb|AAS49166.1| branched-chain alpha-keto aciddecarboxylase (Lactococcus lactis); gi|15004729|ref|NP_149189.11Pyruvate decarboxylase (Clostridium acetobutylicum ATCC 824);gi|82749898|ref|YP_415639.11 probable pyruvate decarboxylase(Staphylococcus aureus RF122); gi|77961217|ref|ZP_00825060.1| COG3961:pyruvate decarboxylase and related thiamine pyrophosphate-requiringenzymes (Yersinia mollaretii ATCC 43969); gi|71065418|ref|YP_264145.1|putative pyruvate decarboxylase (Psychrobacter arcticus 273-4);gi|16761331|ref|NP_456948.1| putative decarboxylase (Salmonella entericasubsp. enterica serovar Typhi str. CT18); gi|93005792|ref|YP_580229.1|pyruvate decarboxylase (Psychrobacter cryohalolentis K5);gi|23129016|ref|ZP_00110850.1|COG3961 pyruvate decarboxylase and relatedthiamine pyrophosphate-requiring enzymes (Nostoc punctiforme PCC 73102);gi|16417060|gb|AAL18557.1|AF354297_1 pyruvate decarboxylase (Sarcinaventriculi); gi|15607993|ref|NP_215368.1| probable pyruvate orindole-3-pyruvate decarboxylase (pdc) (Mycobacterium tuberculosisH37Rv); gi|41406881|ref|NP_959717.1| Pdc (Mycobacterium avium subsp.paratuberculosis K-10); gi|91779968|ref|YP_555176.1| putative pyruvatedecarboxylase (Burkholderia xenovorans LB400);gi|15828161|ref|NP_302424.1| pyruvate (or indolepyruvate) decarboxylase(Mycobacterium leprae TN); gi|118616174|ref|YP_904506.1| pyruvate orindole-3-pyruvate decarboxylase (Pdc) (Mycobacterium ulcerans Agy99);gi|67989660|ref|NP_001018185.1| hypothetical protein SPAC3H8.01(Schizosaccharomyces pombe 972h-); gi|21666011|gb|AAM73540.1| AF282847_1pyruvate decarboxylase PdcB (Rhizopus oryzae);gi|69291130|ref|ZP_00619161.1| pyruvate decarboxylase:pyruvatedecarboxylase (Kineococcus radiotolerans SRS30216);gi|66363022|ref|XP_628477.1| pyruvate decarboxylase (Cryptosporidiumparvum Iowa II); gi|70981398|ref|XP_731481.1| pyruvate decarboxylase(Aspergillus fumigatus Af293); gi|121704274|ref|XP_001270401.1| pyruvatedecarboxylase, putative (Aspergillus clavatus NRRL 1);gi|119467089|ref|XP_001257351.1| pyruvate decarboxylase, putative(Neosartorya fischeri NRRL 181); gi|26554143|ref|NP_758077.1| pyruvatedecarboxylase (Mycoplasma penetrans HF-2);gi|21666009|gb|AAM73539.1|AF282846_1 pyruvate decarboxylase PdcA(Rhizopus oryzae).

Alcohol dehydrogenases (adh) catalyze the final step of amino acidcatabolism, conversion of an aldehyde to a long chain or complexalcohol. Various adh genes are known in the art. As indicated herein,adh1 homologs and variants include, for example, adh2, adh3, adh4, adh5,adh 6 and sfa1 (see, e.g., SFA (Saccharomyces cerevisiae)gi|288591|emb|CAA48161.1| (288591); the sequence associated with theaccession number is incorporated herein by reference).

Citramalate synthase catalyzes the condensation of pyruvate and acetate.CimA encodes a citramalate synthase. Homologs and variants are known andinclude, for example, citramalate synthase (Leptospira biflexa serovarPatoc) gi|116664687|gb|ABK13757.1|(116664687); citramalate synthase(Leptospira biflexa serovar Monteralerio)gi|116664685|gb|ABK13756.1|(116664685); citramalate synthase (Leptospirainterrogans serovar Hebdomadis) gi|116664683|gb|ABK13755.1|(116664683);citramalate synthase (Leptospira interrogans serovar Pomona)gi|116664681|gb|ABK13754.1| (116664681); citramalate synthase(Leptospira interrogans serovar Australis)gi|116664679|gb|ABK13753.1|(116664679); citramalate synthase (Leptospirainterrogans serovar Autumnalis) gi|116664677|gb|ABK13752.1|(116664677);citramalate synthase (Leptospira interrogans serovar Pyrogenes)gi|116664675|gb|ABK13751.1|(116664675); citramalate synthase (Leptospirainterrogans serovar Canicola) gi|116664673|gb|ABK13750.1|(116664673);citramalate synthase (Leptospira interrogans serovar Lai)gi|116664671|gb|ABK13749.1|(116664671); CimA (Leptospira meyeri serovarSemaranga) gi|119720987|gb|ABL98031.1|(119720987); (R)-citramalatesynthase gi|2492795|sp|Q58787.1|CIMA_METJA(2492795); (R)-citramalatesynthase gi|22095547|sp|P58966.1|CIMA_METMA(22095547); (R)-citramalatesynthase gi|22001554|sp|Q8TJJ1.1|CIMA_METAC(22001554); (R)-citramalatesynthase gi|22001553|sp|026819.1|CIMA_METTH(22001553); (R)-citramalatesynthase gi|22001555|sp|Q8TYB1.1|CIMA_METKA(22001555); (R)-citramalatesynthase (Methanococcus maripaludis S2)gi|45358581|ref|NP_988138.1|(45358581); (R)-citramalate synthase(Methanococcus maripaludis S2) gi|44921339|emb|CAF30574.1|(44921339);and similar to (R)-citramalate synthase (Candidatus Kueneniastuttgartiensis) gi|91203541|emb|CAJ71194.1|(91203541), each sequenceassociated with the foregoing accession numbers is incorporated hereinby reference.

It is understood that a range of microorganisms can be modified toinclude a recombinant metabolic pathway suitable for the production ofe.g., 1-propanol, isobutanol, 1-butanol, 2-methyl-1-butanol,3-methyl-1-butanol, or 2-phenylethanol. It is also understood thatvarious microorganisms can act as “sources” for genetic materialencoding target enzymes suitable for use in a recombinant microorganismprovided herein. The term “microorganism” includes prokaryotic andeukaryotic microbial species from the Domains Archaea, Bacteria, andEucarya, the latter including yeast and filamentous fungi, protozoa,algae, or higher Protista. The terms “microbial cells” and “microbes”are used interchangeably with the term “microorganism”.

The term “prokaryotes” is art recognized and refers to cells whichcontain no nucleus or other cell organelles. The prokaryotes aregenerally classified in one of two domains, the Bacteria and theArchaea. The definitive difference between organisms of the Archaea andBacteria domains is based on fundamental differences in the nucleotidebase sequence in the 16S ribosomal RNA.

The term “Archaea” refers to a categorization of organisms of thedivision Mendosicutes, typically found in unusual environments anddistinguished from the rest of the prokaryotes by several criteria,including the number of ribosomal proteins and the lack of muramic acidin cell walls. On the basis of ssrRNA analysis, the Archaea consist oftwo phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota.On the basis of their physiology, the Archaea can be organized intothree types: methanogens (prokaryotes that produce methane); extremehalophiles (prokaryotes that live at very high concentrations of salt((NaCl)); and extreme (hyper) thermophilus (prokaryotes that live atvery high temperatures). Besides the unifying archaeal features thatdistinguish them from Bacteria (i.e., no murein in cell wall,ester-linked membrane lipids, and the like), these prokaryotes exhibitunique structural or biochemical attributes which adapt them to theirparticular habitats. The Crenarchaeota consists mainly ofhyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeotacontains the methanogens and extreme halophiles.

“Bacteria”, or “eubacteria”, refers to a domain of prokaryoticorganisms. Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; and (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or over-express endogenouspolynucleotides, or to express non-endogenous sequences, such as thoseincluded in a vector, or which have a reduction in expression of anendogenous gene. The polynucleotide generally encodes a target enzymeinvolved in a metabolic pathway for producing a desired metabolite asdescribed above. Accordingly, recombinant microorganisms describedherein have been genetically engineered to express or over-expresstarget enzymes not previously expressed or over-expressed by a parentalmicroorganism. It is understood that the terms “recombinantmicroorganism” and “recombinant host cell” refer not only to theparticular recombinant microorganism but to the progeny or potentialprogeny of such a microorganism. E. coli, Yeast, Corynebacterium,Lactobacillus, Bacillus, and the like, are particularly useful as a hostcell.

A “parental microorganism” refers to a cell used to generate, or derive,a recombinant microorganism. The term “parental microorganism” describesa cell that occurs in nature, i.e., a “wild-type” cell that has not beengenetically modified. The term “parental microorganism” also describes acell that has been genetically modified but which does not express orover-express a target enzyme, e.g., an enzyme involved in thebiosynthetic pathway for the production of a desired metabolite such as1-propanol, isobutanol, 1-butanol, 2-methyl-1-butanol,3-methyl-1-butanol, or 2-phenylethanol. For example, a wild-typemicroorganism can be genetically modified to express or over-express afirst target enzyme such as acetolactate synthase. This microorganismcan act as a parental microorganism in the generation of a microorganismmodified to express or over-express a second target enzyme, e.g.,acetohydroxy acid isomeroreductase (IlvC). In turn, the microorganismmodified to express or over-express, e.g., acetolactate synthase andacetohydroxy acid isomeroreductase, can be modified to express orover-express a third target enzyme, e.g., dihydroxy-acid dehydratase(IlvD). Accordingly, a parental microorganism, or host microorganism,functions as a reference cell for successive genetic modificationevents. Each modification event can be accomplished by introducing anucleic acid molecule into the reference cell. The introductionfacilitates the expression or over-expression of a target enzyme. It isunderstood that the term “facilitates” encompasses the activation ofendogenous polynucleotides encoding a target enzyme through geneticmodification of, e.g., a promoter sequence in a parental microorganism.It is further understood that the term “facilitates” encompasses theintroduction of exogenous polynucleotides encoding a target enzyme intoa parental microorganism.

In another embodiment, a method of producing a recombinant microorganismthat converts a suitable carbon substrate to, e.g., isobutanol, 2-methyl1-butanol, and/or 3-methyl 1-butanol is provided. The method includestransforming a microorganism with one or more recombinantpolynucleotides encoding polypeptides that include, for example, anenzyme having both acetolatate synthase and 2-ketodecarboxylase activity(e.g., als), and a polypeptide having alcohol dehydrogenase activity.Polynucleotides that encode enzymes useful for generating metabolitesincluding homologs, variants, fragments, related fusion proteins, orfunctional equivalents thereof, are used in recombinant nucleic acidmolecules that direct the expression of such polypeptides in appropriatehost cells, such as bacterial or yeast cells. It is understood that theaddition of sequences which do not alter the encoded activity of apolynucleotide, such as the addition of a nonfunctional or non-codingsequence, is a conservative variation of the basic nucleic acid. The“activity” of an enzyme is a measure of its ability to catalyze areaction resulting in a metabolite, i.e., to “function”, and may beexpressed as the rate at which the metabolite of the reaction isproduced. For example, enzyme activity can be represented as the amountof metabolite produced per unit of time or per unit of enzyme (e.g.,concentration or weight), or in terms of affinity or dissociationconstants.

It is understood that the polynucleotides described above include“genes” and that the nucleic acid molecules described above include“vectors” or “plasmids.” For example, a polynucleotide encoding apolypeptide having acetolactate synthase and 2-ketodecarboxylaseactivity can be encoded by an als gene or homolog thereof. Accordingly,the term “gene”, also called a “structural gene” refers to apolynucleotide that codes for a particular sequence of amino acids,which comprise all or part of one or more proteins or enzymes, and mayinclude regulatory (non-transcribed) DNA sequences, such as promotersequences, which determine, for example, the conditions under which thegene is expressed. The transcribed region of the gene may includeuntranslated regions, including introns, 5′-untranslated region (UTR),and 3′-UTR, as well as the coding sequence. The term “nucleic acid” or“recombinant nucleic acid” refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein results from transcription andtranslation of the open reading frame sequence.

The term “operon” refers to two or more genes which are transcribed as asingle transcriptional unit from a common promoter. In some embodiments,the genes comprising the operon are contiguous genes. It is understoodthat transcription of an entire operon can be modified (i.e., increased,decreased, or eliminated) by modifying the common promoter.Alternatively, any gene or combination of genes in an operon can bemodified to alter the function or activity of the encoded polypeptide.The modification can result in an increase in the activity of theencoded polypeptide. Further, the modification can impart new activitieson the encoded polypeptide. Exemplary new activities include the use ofalternative substrates and/or the ability to function in alternativeenvironmental conditions.

A “vector” is any means by which a nucleic acid can be propagated and/ortransferred between organisms, cells, or cellular components. Vectorsinclude viruses, bacteriophages, pro-viruses, plasmids, phagemids,transposons, and artificial chromosomes such as YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes), and PLACs (plantartificial chromosomes), and the like, that are “episomes,” that is,that replicate autonomously or can integrate into a chromosome of a hostcell. A vector can also be a naked RNA polynucleotide, a naked DNApolynucleotide, a polynucleotide composed of both DNA and RNA within thesame strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugatedDNA or RNA, a liposome-conjugated DNA, or the like, that are notepisomal in nature, or it can be an organism which comprises one or moreof the above polynucleotide constructs such as an agrobacterium or abacterium.

“Transformation” refers to the process by which a vector is introducedinto a host cell. Transformation (or transduction, or transfection) canbe achieved by any one of a number of means including electroporation,microinjection, biolistics (or particle bombardment-mediated delivery),or agrobacterium mediated transformation.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given amino acidsequence (e.g., enzyme) of the disclosure. The native DNA sequenceencoding the biosynthetic enzymes described herein are referenced hereinmerely to illustrate an embodiment of the disclosure, and the disclosureincludes DNA compounds of any sequence that encode the amino acidsequences of the polypeptides and proteins of the enzymes utilized inthe methods of the disclosure. In similar fashion, a polypeptide cantypically tolerate one or more amino acid substitutions, deletions,and/or insertions in its amino acid sequence without loss or significantloss of a desired activity. The disclosure includes such polypeptideswith different amino acid sequences than the specific proteins describedherein so long as the modified or variant polypeptides have theenzymatic anabolic or catabolic activity of the reference polypeptide,and the amino acid sequences encoded by the DNA sequences shown hereinmerely illustrate embodiments of the disclosure.

The disclosure provides nucleic acid molecules in the form ofrecombinant DNA expression vectors or plasmids, as described in moredetail below, that encode one or more target enzymes. Generally, suchvectors can either replicate in the cytoplasm of the host microorganismor integrate into the chromosomal DNA of the host microorganism. Ineither case, the vector can be a stable vector (i.e., the vector remainspresent over many cell divisions, even if only with selective pressure)or a transient vector (i.e., the vector is gradually lost by hostmicroorganisms with increasing numbers of cell divisions). Thedisclosure provides DNA molecules in isolated (i.e., not pure, butexisting in a preparation in an abundance and/or concentration not foundin nature) and purified (i.e., substantially free of contaminatingmaterials or substantially free of materials with which thecorresponding DNA would be found in nature) forms.

Provided herein are methods for the heterologous expression of one ormore of the biosynthetic genes involved in alcohol production (e.g.,isobutanol production or biosynthesis and recombinant DNA expressionvectors useful in the method. Thus, included within the scope of thedisclosure are recombinant expression vectors that include such nucleicacids. The term “expression vector” refers to a nucleic acid that can beintroduced into a host microorganism or cell-free transcription andtranslation system. An expression vector can be maintained permanentlyor transiently in a microorganism, whether as part of the chromosomal orother DNA in the microorganism or in any cellular compartment, such as areplicating vector in the cytoplasm. An expression vector also comprisesa promoter that drives expression of an RNA, which typically istranslated into a polypeptide in the microorganism or cell extract. Forefficient translation of RNA into protein, the expression vector alsotypically contains a ribosome-binding site sequence positioned upstreamof the start codon of the coding sequence of the gene to be expressed.Other elements, such as enhancers, secretion signal sequences,transcription termination sequences, and one or more marker genes bywhich host microorganisms containing the vector can be identified and/orselected, may also be present in an expression vector. Selectablemarkers, i.e., genes that confer antibiotic resistance or sensitivity,are used and confer a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium.

The various components of an expression vector can vary widely,depending on the intended use of the vector and the host cell(s) inwhich the vector is intended to replicate or drive expression.Expression vector components suitable for the expression of genes andmaintenance of vectors in E. coli, yeast, Streptomyces, and othercommonly used cells are widely known and commercially available. Forexample, suitable promoters for inclusion in the expression vectors ofthe disclosure include those that function in eukaryotic or prokaryotichost microorganisms. Promoters can comprise regulatory sequences thatallow for regulation of expression relative to the growth of the hostmicroorganism or that cause the expression of a gene to be turned on oroff in response to a chemical or physical stimulus. For E. coli andcertain other bacterial host cells, promoters derived from genes forbiosynthetic enzymes, antibiotic-resistance conferring enzymes, andphage proteins can be used and include, for example, the galactose,lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla),bacteriophage lambda PL, and T5 promoters. In addition, syntheticpromoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can alsobe used. For E. coli expression vectors, it is useful to include an E.coli origin of replication, such as from pUC, p1P, p1, and pBR.

A nucleic acid of the disclosure can be amplified using cDNA, mRNA, oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques and those procedures described in the Examples section below.The nucleic acid so amplified can be cloned into an appropriate vectorand characterized by DNA sequence analysis. Furthermore,oligonucleotides corresponding to nucleotide sequences can be preparedby standard synthetic techniques, e.g., using an automated DNAsynthesizer.

In another embodiment, a method for producing an alcohol, e.g.,isobutanol, is provided. The method includes culturing a recombinantmicroorganism as provided herein in the presence of a suitable substrateand under conditions suitable for the conversion of the substrate toisobutanol. The alcohol produced by a microorganism provided herein canbe detected by any method known to the skilled artisan. Such methodsinclude mass spectrometry. Culture conditions suitable for the growthand maintenance of a recombinant microorganism provided herein aredescribed in the Examples below. The skilled artisan will recognize thatsuch conditions can be modified to accommodate the requirements of eachmicroorganism.

As previously discussed, general texts which describe molecularbiological techniques useful herein, including the use of vectors,promoters and many other relevant topics, include Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152,(Academic Press, Inc., San Diego, Calif.) (“Berger”); Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N. Y., 1989 (“Sambrook”); andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 1999)(“Ausubel”). Examples of protocols sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR), the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of thedisclosure are found in Berger, Sambrook, and Ausubel, as well as inMullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990)PCR Protocols: A Guide to Methods and Applications (Academic Press Inc.San Diego, Calif.) (“Innis”); Arnheim & Levinson (Oct. 1, 1990)C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; Kwoh et al.(1989) Proc. Natl Acad. Sci. USA 86:1173; Guatelli et al. (1990) Proc.Nat'l Acad. Sci. USA 87:1874; Lomell et al. (1989) J. Clin. Chem.35:1826; Landegren et al. (1988) Science 241:1077-1080; Van Brunt (1990)Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560; Barringer etal. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology13:563-564. Improved methods for cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods for amplifying large nucleic acids by PCR are summarized inCheng et al. (1994) Nature 369:684-685 and the references cited therein,in which PCR amplicons of up to 40 kb are generated. One of skill willappreciate that essentially any RNA can be converted into a doublestranded DNA suitable for restriction digestion, PCR expansion, andsequencing using reverse transcriptase and a polymerase. See, e.g.,Ausubel, Sambrook and Berger, all supra.

Appropriate culture conditions are conditions of culture medium pH,ionic strength, nutritive content, and the like; temperature;oxygen/CO₂/nitrogen content; humidity; and other culture conditions thatpermit production of the compound by the host microorganism, i.e., bythe metabolic action of the microorganism. Appropriate cultureconditions are well known for microorganisms that can serve as hostcells.

The invention is illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

Examples

Restriction enzymes and Antarctic phosphatase were from New EnglandBiolabs (Ipswich, Mass.). The rapid DNA ligation kit was from Roche(Mannheim, Germany). KOD DNA polymerase was from EMD Chemicals (SanDiego, Calif.). Oligonucleotides were from Operon (Huntsville, Ala.).

Strains and plasmids. A list of many of the strains, plasmids, andoligos used is given in Table 1. JCL16 (Atsumi et al., (2008) Metab.Eng. 10:305-311) is BW25113 (rrnB_(T14) ΔlacZ_(WJ16) hsdR514ΔaraBAD_(AH33) ΔrhaBAD_(LD78)) (Datsenko and Wanner, (2000) Proc. NatlAcad. Sci. USA 97:6640-6645) with F′ transduced from XL-1 Blue to supplylacI^(q). JCL260 is JCL16 ΔadhE Δfnr-ldhA ΔfrdBC ΔpflB Δpta. The ilvCgene was inactivated by P1 transduction with JW3747 (Baba et al., (2006)Mol. Syst. Biol. 2:2006.0008, doi:10.1038/msb4100050).

TABLE 1 Strains, plasmids and oligos used in the ExamplesRelevant Genotype Reference Strain BW25113 rrnB_(T14) ΔlacZWJ16 (A)hsdR514 ΔaraBAD_(AH33) ΔrhaBAD_(LD78) JCL16 BW25113/F′ [traD36 (B)proAB⁺, lacI^(q) ZΔM15] JCL260 Same as JCL16 but (C) ΔadhE Δfnr-ldhAΔfrdBC ΔpflB Δpta SA296 Same as JCL260 This work but ΔilvC KS145Same as JCL16 but (D) ΔilvI ΔilvB Plasmid pSA55 ColE1 ori; Amp^(r); (C)P_(L) lacO₁::kivd-ADH2 pSA69 p15A ori; Kan^(r); (C) P_(L) lacO₁::alsS-ilvC-ilvD pCS27 p15A ori; Kan^(r); (E) P_(L) lacO₁::MCS1 pZL8p15A ori; Kan^(R); This work P_(L) lacO₁::alsS pSA159 Derivative of This work pPETDuet-1 with alsS pSA166 Derivative of This workpPETDuet-1 with alsS (Q487A) pSA187 Derivative of This workpETDUET-1 with alsS(Q487I) pSA188 Derivative of This work pETDUET-1 withalsS(Q487S) pSA205 Derivative of This work pETDUET-1 with alsS(Q487G)pSA206 Derivative of This work pETDUET-1 with alsS(Q487L) OligoSequence 5′ → 3′ A124 ACGCAGTCGACCTAGA (C) GAGCTTTCGTTTTCAT GAGT(SEQ ID NO: 11) A297 CGGGATCCGTTGACAA This work AAGCAACAAAAGAACA AA(SEQ ID NO: 12) A298 ACGCAGTCGACCTAGA This work GAGCTTTCGTTTTCAT GAGT(SEQ ID NO: 13) A300 AATAAGACGTCTAAGA This work AACCATTATTATCATG(SEQ ID NO: 14) A305 GAATGCAACCATGTCA This work TATGTGCTG(SEQ ID NO: 15) A306 ATGGAACGACAGCACA This work TATGACATGGTTGCATTCAACCAATTGAAAAA A TATAACCGTAC (SEQ ID NO: 16) A307 ATGGAACGACAGCACAThis work TATGACATGGTTGCAT TCGCCCAATTGAAAAA A TATAACCGTAC SEQ ID NO: 17)

REFERENCES

-   (A) Datsenko and Wanner, (2000) Proc. Natl Acad. Sci. USA    97:6640-6645; (B) Atsumi et al., (2008) Metab. Eng. 10:305-311; (C)    Atsumi et al., (2008) Nature 451:86-89; (D) Atsumi and Liao, (2008)    Appl. Environ. Microbiol. 74:7802-78081; (E) Shen and Liao, (2008)    Metab. Eng. 10:312-320.

To clone alsS, pSA69 (Atsumi et al., (2008) Nature 451:86-89) wasdigested with AatII and SalI. A shorter fragment was purified and clonedinto plasmid pCS27 (Shen and Liao, (2008) Metab. Eng. 10:312-320) cutwith the same enzymes, creating pZL8. Both alsS single site mutations(Q487N and Q487A) were introduced using PCR directed mutagenesis. Tointroduce the mutation into alsS, pSA69 was used as PCR template withA306 and A124 (Q487N) and A307 and A124 (Q487A). The beginning of thealsS gene located on pSA69 was also amplified from the AatII siteupstream of the ribosome binding site to the 1,485th base in the alsSgene, using primers A300 and A305. These two fragments were then joinedby splice overlap extension (SOE). The products were digested with AatIIand SalI and cloned into pSA69 cut with the same enzyme, creating pSA163and pSA164.

For protein over-expression and purification, the wild-type and alsSvariants were amplified with primers A297 and A298. PCR products weredigested with BamHI and SalI and cloned into pETDuet-1 (Novagen(Madison, Wis.)) cut with the same enzymes (Table 2), creating pS159 andpSA166.

TABLE 2 Kinetic parameters of the wild-type AlsS (B. subtilis) and thevariants for acetolactate synthase and decarboxylase activity^(a) Aminoacid at Pyruvate KIV residue 487 K_(m) (mM) k_(cat) (s⁻¹) k_(cat)/K_(m)ratio K_(m) (mM) k_(cat) (s⁻¹) k_(cat)/K_(m) ratio Q 13.6 ± 0.8  121 ±13 8.9 ± 1.1 300 ± 35 8.9 ± 1.2  0.03 ± 0.005 A 8.7 ± 0.5 58 ± 8 6.7 ±1.0 186 ± 27 1.1 ± 0.5 0.006 ± 0.003 G 1.6 ± 0.4 11 ± 5 6.9 ± 3.6 175 ±18 0.8 ± 0.2 0.005 ± 0.001 S 1.1 ± 0.6 11 ± 6  10 ± 7.7 154 ± 21 0.8 ±0.3 0.005 ± 0.002 L ND ND ND 342 ± 45 5.4 ± 0.9  0.02 ± 0.003 I ND ND ND323 ± 26 4.8 ± 0.5  0.01 ± 0.002 ^(a)The values shown after the ± signsare standard deviations. ND, not detectable.

Medium and culture conditions for isobutanol production. M9 medium (64 gNa₂HPO₄.7H₂O, 15 g KH₂PO₄, 2.5 g NaCl, 5 g NH₄Cl, 2 mM MgSO₄, 0.1 mMCaCl₂, 10 mg thiamine per liter water) containing 36 g/L glucose, 5 g/Lyeast extract, 100 μg/ml ampicillin, 30 μg/ml kanamycin and 1 ml/literof Trace Metal Mix A5 (2.86 g H₃BO₃, 1.81 g MnCl₂.4H₂O, 0.222 gZnSO₄.7H₂O, 0.39 g Na₂MoO₄.2H₂O, 0.079 g CuSO₄.5H₂O, 49.4 mgCo(NO₃)₂.6H₂O per liter water) was used for cell growth. Preculture intest tubes containing 3 ml of medium was performed at 37° C. overnighton a rotary shaker (250 rpm). Overnight culture was diluted 1:100 into20 ml of fresh medium in a 250 ml screw cap conical flask. Cells weregrown at 37° C. for 3 hrs, followed by adding 0.1 mM IPTG(isopropyl-β-D-thiogalactopyranoside). Production was performed undermicroaerobic conditions at 30° C. on a rotary shaker (250 rpm) for 24hrs. Isobutanol was quantified by a gas chromatographyflame ionizationdetector as previously described (Atsumi et al., (2008) Nature451:86-89). Secreted pyruvate was quantified by high-performance liquidchromatography as previously described (Atsumi et al., (2008) Metab.Eng. 10:305-311).

Protein purification. The wild type AlsS and AlsS variants weresynthesized from a His-tag plasmid in E. coli strain BL21 Star™ (DE3)(Invitrogen (Carlsbad, Calif.)) followed by purification withNi-nitrilotriacetic acid (NTA) spin columns (Qiagen (Valencia, Calif.)).Protein concentrations were determined by the Bradford assay (Bio-Rad(Hercules, Calif.)).

Als enzyme activity. An enzyme assay for Als activity of AlsS wascarried out in 1 ml of morpholinepropanesulfonic acid (MOPS) buffer (pH7.0) containing 80 nM AlsS, 100 mM MOPS (pH 7.0), 1 mM MgCl₂, 0.1 mMTPP, 10 mM acetate, and various concentrations of pyruvate at 37° C. for10 min. The reaction was terminated by acidification of the solutionwith 0.1 ml of 50% H₂SO₄. The mixture was incubated for an additional 25min at 37° C. to allow for the acid hydrolysis of the acetolactate toacetoin. Acetoin formation was measured as described previously(Holtzclaw and Chapman, (1975) J. Bacteriol. 121:917-922). One unit ofenzyme activity was defined as the amount of enzyme that converts 1 μmolof substrate into product in 1 minute under these conditions. The K_(m)values for pyruvate and the V_(max) were extrapolated after nonlinearregression of the experimental points with the Gauss-Newton method usingMatlab.

Kdc activity assay. The enzyme assay for Kdc activity of AlsS wascarried out in reaction mixtures containing 80 nM AlsS, 100 mM MOPS (pH7.0), 1 mM MgCl₂, 0.1 mM TPP, 10 mM acetate, and various concentrationsof KIV at 37° C. for 1 h. The production of isobutyraldehyde wasconfirmed to be linear over 1 h. Isobutyraldehyde was measured by a gaschromatography-flame ionization detector as previously described (Atsumiet al., (2008) Nature 451:86-89). One unit of enzyme activity wasdefined as the amount of enzyme that converts 1 μmol of substrate intoproduct in 1 minute under these conditions. The K_(m) values for KIV andthe V_(max) were extrapolated after nonlinear regression of theexperimental points with the Gauss-Newton method using Matlab.

TABLE 3 Sequences of included sequence listing. SEQ ID NO: Comment 1 DNAsequence of acetolactate synthase (B. subtilis) 2 Protein sequence ofacetolactate synthase (B. subtilis) 3 DNA sequence of acetolactatesynthase (B. subtilis) where the first g is changed to a 4 Proteinsequence of acetolacate synthase (B. subtilis) where the first aminoacid is M instead of V 5 DNA sequence of acetolactate synthase Q487Nmutant (mutant of SEQ ID NO: 3) 6 Protein sequence of acetolactatesynthase Q487N mutant (mutant of SEQ ID NO: 5) 7 Protein sequence ofacetolactate synthase Q487N mutant (mutant of SEQ ID NO: 2) 8 DNAsequence of acetolactate synthase Q487A mutant (mutant of SEQ ID NO: 1)9 Protein sequence of acetolactate synthase Q487A mutant (mutant of SEQID NO: 4) 10 Protein sequence of acetolactate synthase Q487A mutant(mutant of SEQ ID NO: 2) 11 See Table 1 12 See Table 1 13 See Table 1 14See Table 1 15 See Table 1 16 See Table 1 17 See Table 1

Analysis of the Kdc-independent isobutanol production. Since E. colidoes not have any Kdc, it was hypothesized that pyruvate dehydrogenase(PDH) or 2-ketoglutarate dehydrogenase (KGDH) of E. coli could catalyzethe conversion of KIV to isobutyryl coenzyme A, followed by theconversion of isobutyryl coenzyme A to isobutyraldehyde and thenisobutanol by aldehyde and alcohol dehydrogenases. To test thesepossibilities, aceE and sucA were deleted, which encode subunits of thePDH and KGDH complexes, respectively. However, this double knockoutstrain with over-expression of alsS, ilvC, and ilvD was still capable ofproducing isobutanol, indicating that neither PDH nor KGDH catalyzes thereaction in the Kdc-independent isobutanol production.

To determine essential components for the Kdc-independent isobutanolproduction, isobutanol production from the strain over-expressingdifferent combinations of alsS, ilvC, and ilvD was measured (FIG. 2).The strain over-expressing alsS alone produced isobutanol, but thestrain over-expressing ilvC and ilvD did not (FIG. 2A). When ilvC andilvD were over-expressed with alsS, isobutanol production increasednearly ninefold (FIG. 2A). Because the only known activity of AlsS isacetolactate synthase, it is unclear how the strain could produceisobutanol only with alsS over-expression. As a control experiment, ilvIand ilvH (E. coli), which encodes an acetohydroxy acid synthase (Ahas)instead of AlsS (B. subtilis), were over-expressed. The strainover-expressing ilvI and ilvH (E. coli) did not produce isobutanol.Increasing AlsS levels in E. coli led to a parallel increase in theformation of acetoin, which is the product of spontaneousdecarboxylation of 2-acetolactate (Aristidou et al., (1994) Biotechnol.Bioeng. 44:944-951). To test whether some enzymes in E. coli couldutilize acetoin as a substrate for isobutanol production, acetoin wasfed to the E. coli culture. Neither isobutyraldehyde nor isobutanol wasdetected from this culture, indicating that acetoin was not a precursorof isobutanol in this pathway. To confirm that the Kdc-independentpathway used the same route as the Kdc-dependent pathway, the ilvC geneon the genome was deleted (FIG. 2A). The deletion of ilvC abolishedisobutanol production, indicating that this Kdc-independent pathwayutilized KIV as a precursor (FIG. 2A).

KIV was then supplied to the medium to assess the capability to utilizeKIV for isobutanol production (FIG. 2B). The strain without AlsS did notproduce isobutanol in the presence of 6 g/liter of KIV (FIG. 2B), butaddition of KIV to strains over-expressing alsS led to isobutanolproduction in the wild-type and ΔilvC backgrounds (FIG. 2B). These testsrevealed that Kdc-independent isobutanol production requiresover-expression of alsS and a high concentration of KIV. Feeding of KIVto the strain over-expressing alsS, ilvC, and ilvD did not change theproduction of isobutanol (FIG. 2C), presumably because the concentrationof KIV may already saturate the enzyme which utilizes KIV for isobutanolproduction or the efficiency of KIV uptake may decrease (FIG. 2C).

Characterization of wild type and AlsS variants. It was hypothesizedthat AlsS could catalyze the decarboxylation of KIV and giveisobutyraldehyde without the nucleophilic attack of the second pyruvatein the presence of a high concentration of KIV and a low concentrationof pyruvate. Because the over-expression of alsS, ilvC, and ilvDsignificantly decreases the secretion of pyruvate to below a detectionlimit of 0.1 mM (the host strain without these plasmids secretes 7 mMpyruvate), it is possible that under this condition, KIV reacts with TPPand undergoes decarboxylation, and then escapes by givingisobutyraldehyde before undergoing the carboligation.

Purification and characterization of wild type and AlsS variants. Usingthe crystal structure of Klebsiella pneumoniae AlsS, Pang et al., (2004)J. Biol. Chem. 279:2242-2253) showed that the extended side chain ofGln483 causes steric hindrance with the larger substrate,2-ketobutyrate, which explains why AlsS reacts very poorly with a larger2-keto acid as the second substrate (Gollop et al., (1990) J. Bacteriol.172:344-3449; Pang et al., (2004) J. Biol. Chem. 279:2242-2253). Gln483in K. pneumoniae AlsS has been shown to be in close proximity to thefirst pyruvate and also involved in second-substrate specificity (Panget al., (2004) J. Biol. Chem. 279:2242-2253), suggesting that Gln483 mayplay a role in the release of aldehydes (see FIG. 3A). The residuecorresponding to Gln483 in K. pneumoniae AlsS is Gln487 in B. subtilisAlsS. To test whether Gln487 could play a role in decarboxylation,Gln487 was replaced with various other amino acids. For example, Gln487was replaced with Asn and Ala. The side-chain of Asn has an amine grouplike Gln but it is shorter than that of Gln. The side-chain of Ala isshorter than that of Gln but does not contain an amine group. These AlsSmutants were over-expressed with IlvC and IlvD. Isobutanol productionwith AlsS (Q487N) and AlsS (Q487A) decreased by 25% and 90%,respectively (FIG. 3B). The replacement of Q487 with alanine nearlyabolished isobutanol production, indicating that this residue isimportant for either acetolactate synthase or 2-ketoisovaleratedecarboxylase activity. To test which reactions were affected by thesemutations, KDC was over-expressed (FIG. 3C). With over-expression ofKDC, all strains produced similar levels of isobutanol (FIG. 3C),indicating the replacement of Q487 only affected the decarboxylaseactivity.

Purification and characterization of wild type and AlsS variants. Toassay the Kdc activity (FIG. 1C, bottom) of AlsS, His-tagged wild-typeAlsS was expressed from a His-tag plasmid and purified as describedabove. The Kdc activity of the His-tagged wild-type AlsS was 5.5μmol·min⁻¹·mg⁻¹, while isobutyraldehyde production was not detected froma negative control experiment without AlsS. Although the activity wasweak, AlsS showed the decarboxylase activity toward KIV in vitro.

The kinetic parameters were measured for AlsS variants (Table 2). Thek_(cat)/K_(m) values for pyruvate of Q487 variants with small residues(Q487A, Q487G, and Q487S) were similar to that of the wild type, whilethe k_(cat)/K_(m) values for KIV of these variants decreaseddramatically (Table 2). Q487L and Q487I replacements impaired Alsactivity (Table 2). However, the k_(cat)/K_(m) values for KIV of thesevariants were similar to that of the wild type. The wild type and allvariants showed extremely high K_(m) values for KIV (Table 2), which mayexplain why an increase of the flux toward KIV is required for thedecarboxylase activity of AlsS.

Effects of Q487 replacements for isobutanol production. To test theseAlsS variants' capability to produce isobutanol, which requires both Als(FIG. 1C, top) and Kdc (FIG. 1C, bottom) activities, these alsS variantswere over-expressed with ilvC and ilvD. Isobutanol production with AlsS(Q487N) was similar to the production achieved using wild-type AlsS(FIG. 4A), presumably because the side chain of Asn has an amine group,like Gln. However, the replacement of Q487 with valine, alanine,glycine, serine, leucine, and isoleucine nearly abolished isobutanolproduction (FIG. 4A). According to the results of the performed enzymeassays (Table 2), the replacement of Q487 with glycine and serinemaintained Als activity and decreased Kdc activity. The ratios of K_(m)for KIV to K_(m) for pyruvate of Q487G and Q487S were 109±30 and 140±79,respectively, while the ratio for the wild-type enzyme was 20±2.9. Thestrains with either Q487G or Q487S cannot produce isobutanol, presumablybecause the Kdc activity of Q487G and Q487S could not compete with theAls activity. Enzyme assays showed that Q487L and Q487I replacementsimpaired Als activity. Increased flux toward KIV was found to beimportant for isobutanol production when using AlsS for Kdc activity(FIGS. 2A and 2B), but the K_(m) values for KIV of Q487L and Q487I were342±45 mM and 323±26 mM, respectively (Table 2), which were extremelyhigh. Because these K_(m) values are extremely high, the strains withthese replacements could not produce isobutanol, presumably because theintracellular concentration of KIV would not be high enough for the Kdcactivity. No replacements were found that could increase isobutanolproduction.

Effects of Q487 replacements for Als activity. To distinguish betweenAls and Kdc activities in these variants, the growth rate of an E. colistrain KS145 (ΔilvI ΔilvB) expressing various Q487 variants was testedin a minimum glucose medium supplemented with L-isoleucine. AlsS is adistant homologue of Ahas, which is responsible for both Als (FIG. 1C,top) and 2-aceto-2-hydroxy butyrate synthase (Ahbs) (FIG. 1C, middle)activities in the branched chain amino acid biosynthesis. The KS145strain does not have Als and Ahbs activities (FIG. 1C); thus, thespecific growth rate in the minimal medium with L-isoleucine reflectsthe Als activity. FIG. 4B shows that all of the Q487 variants retainsignificant Als activity. Considering that most of the Q487 variants didnot generate isobutanol (FIG. 4A), it was concluded that AlsS is indeedresponsible for the Kdc activity observed in isobutanol synthesis, andthat Q487 is important for this activity. KS145 cells expressing Q487Lor Q487I showed slow growth with the L-isoleucine supplement (FIG. 4B),indicating that these replacements would reduce Als activity. Theseresults were consistent with the results of enzyme assays (Table 2). Inthe structure model of K. pneumoniae AlsS, the C-1 carbonyl oxygen ofthe modeled second pyruvate is hydrogen-bonded to the side chain ofGln483 (Pang et al., (2004) J. Biol. Chem. 279:2242-2253). Thus, thenonpolar side chain of isoleucine and leucine in the 487th residue wouldreduce the binding affinity of the second pyruvate to the site. Nogrowth phenotype was observed in any strain while grown on minimumglucose medium supplemented with L-valine, L-leucine, and L-isoleucine.

Effects of Q487 replacements for Ahbs activity. AlsS reacts very poorlywith the larger substrate, 2-ketobutyrate (Gollop et al., (1990) J.Bacteriol. 172:3444-3449). If these replacements change the secondsubstrate specificity by removing steric hindrance, the AlsS variantswould gain the Ahbs activity so that the KS145 cells expressing thevariants could grow in minimal medium with L-valine and L-leucinesupplementation. As predicted, KS145 cells expressing the wild-type AlsSwere unable to grow without L-isoleucine (FIG. 4C), indicating that thewild-type AlsS was not capable of catalyzing Ahbs reaction, which isconsistent with previous studies (Huseby and Stormer, (1971) Eur. J.Biochem. 20:215-217). Interestingly, the AlsS variants which containsmall residues (Ala, Gly, and Ser) at the 487th residue rescued thegrowth of KS145 under the same conditions (FIG. 4C). This resultsuggests that the replacement of Q487 with small side chain amino acidswould make the substrate binding site larger so that the variants areable to react with 2-ketobutyrate as the second substrate (Ahbsactivity) (FIG. 1C, middle).

The foregoing evidence shows that Kdc is not essential for isobutanolproduction and that AlsS, previously known only to have Als activity,can catalyze the decarboxylation of KIV. The use of mutational studiesallowed the inventors to identify that Q487 is important for Kdcactivity. The disclosure also demonstrates that AlsS is able to functionas a KDC to release aldehydes after decarboxylation. KDC requires onlyone substrate binding event and one catalytic reaction, while ALSrequires two substrate binding events and two catalytic reactions(decarboxylation and ligation using the second substrate). Since KDC issimpler than ALS, KDC may have arisen earlier than ALS.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the devices, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A recombinantmicroorganism that produces isobutanol wherein the alcohol is producedfrom a metabolite comprising 2-keto acid and wherein the organismcomprises (i) a heterologous mutant acetolactate synthase lacking2-ketoacid decarboxylase activity and (ii) a heterologous 2-ketoaciddecarboxylase.
 2. The recombinant microorganism of claim 1, wherein themicroorganism is an E. coli.
 3. The recombinant microorganism of claim1, wherein the acetolactate synthase is derived from B. subtilis.
 4. Therecombinant microorganism of claim 3, wherein the acetolactate synthasecomprises a sequence that is at least 80%, 90%, 95%, 98%, or 99%identical to SEQ ID NO:2.
 5. The recombinant microorganism of claim 3,wherein the acetolactate synthase comprises a sequence that is at least80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:7.
 6. The recombinantmicroorganism of claim 3, wherein the acetolactate synthase comprisesSEQ ID NO:2.
 7. The recombinant microorganism of claim 3, wherein theacetolactate synthase comprises SEQ ID NO:7.
 8. The recombinantmicroorganism of claim 1, wherein the microorganism is from a genus ofCorynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter,Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus,Serratia, Shigella, Klebsiella, Citrobacter, Saccharomyces, Dekkera,Klyveromyces, or Pichia.
 9. The recombinant microorganism of claim 8,wherein the microorganism is derived from E. coli or S. cerevisiae. 10.The recombinant microorganism of claim 1, wherein the biosyntheticpathway for the production of an amino acid in the organism is modifiedfor production of isobutanol.
 11. The recombinant microorganism of claim1, wherein the microorganism comprises reduced ethanol productioncapability compared to a parental microorganism.
 12. The recombinantmicroorganism of claim 11, wherein the recombinant microorganismcomprises a reduction of an ethanol dehydrogenase, thereby providing areduced ethanol production capability as compared to a parentalmicroorganism.
 13. A method for producing isobutanol, the methodcomprising: viding a recombinant microorganism of claim 1; (b) culturingthe microorganism of (a) in the presence of a suitable substrate ormetabolic intermediate and under conditions suitable for the conversionof the substrate to isobutanol; and (c) substantially purifying theisobutanol.